BSL3 polypeptides

ABSTRACT

The present invention provides nucleic acids encoding B7-related factors that modulate the activation of immune or inflammatory response cells, such as T-cells. Also provided are expression vectors and fusion constructs comprising nucleic acids encoding B7-related polypeptides, including BSL1, BSL2, and BSL3. The present invention further provides isolated B7-related polypeptides, isolated fusion proteins comprising B7-related polypeptides, and antibodies that are specifically reactive with B7-related polypeptides, or portions thereof. In addition, the present invention provides assays utilizing B7-related nucleic acids, polypeptides, or peptides. The present invention further provides compositions of B7-related nucleic acids, polypeptides, fusion proteins, or antibodies that are useful for the immunomodulation of a human or animal subject.

RELATED APPLICATIONS

This application is a divisional application of non-provisionalapplication U.S. Ser. No. 12/701,119, filed Feb. 5, 2010, now U.S. Pat.No. 8,080,636 which is a divisional application of non-provisionalapplication U.S. Ser. No. 11/900,026, filed Sep. 6, 2007, now U.S. Pat.No. 7,723,479, which is a divisional application of non-provisionalapplication U.S. Ser. No. 10/994,824, filed Nov. 22, 2004, now U.S. Pat.No. 7,279,567, which claims benefit to non-provisional application U.S.Ser. No. 09/875,338, filed Jun. 6, 2001, now U.S. Pat. No. 6,965,018,which claims benefit to provisional application U.S. Ser. No.60/209,811, filed Jun. 6, 2000, and provisional application U.S. Ser.No. 60/272,107, filed Feb. 28, 2001, which are incorporated by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to isolated nucleic acids encodingB7-related polypeptides, including BSL1, BSL2, and BSL3, which modulatecells that are important for immune and inflammatory responses, such asT-cells. Also related are expression vectors and fusion constructscomprising nucleic acids encoding B7-related polypeptides. The presentinvention further relates to isolated B7-related polypeptides, isolatedfusion proteins comprising B7-related polypeptides, and antibodies thatare specifically reactive with B7-related polypeptides, or portionsthereof. In addition, the present invention relates to methods ofisolating and identifying the corresponding counter-receptor(s) of theB7-related polypeptides, utilizing B7-related polypeptides, or fusionproteins. Also related are methods of immunomodulation of a subject bythe administration of compositions of the B7-related polypeptides,fusion proteins, cognate antibodies, or portions or derivatives thereof.The present invention further relates to methods of immunomodulation ofanimal or human subjects by the administration of compositions ofgenetically engineered vectors comprising the B7-related polypeptideexpression cassettes as disclosed herein.

BACKGROUND OF THE INVENTION

The primary response of T-cells, involving T-cell activation, expansion,and differentiation is essential for the initiation of an immuneresponse to a foreign antigen. The activation of T-cells by antigenpresenting cells (APCs) requires at least two separate signals (K. E.Hellstrom et al. (1996) Cancer Chemother. Pharmacol. 38:S40-1; N. K.Damle et al. (1992) J. Immunol. 148:1985-92; J. M. Green et al. (1994)Eur. J. Immunol. 24:265-72; E. C. Guinan et al. (1994) Blood 84:3261-82;J. W. Hodge et al. (1995) Cancer Res. 55:3598-603). The first signalcauses T-cell entry into the cell cycle, and is mediated by foreignantigens presented by the major histocompatibility complex (MHC). Thesecond signal, termed costimulation, causes cytokine production andT-cell proliferation, but is neither antigen-specific, nor MHCrestricted (R. H. Schwartz (1990) Science 248:1349-1356).

Costimulation is believed to be mediated by one or more distinct cellssurface molecules expressed by APCs (M. K. Jenkins et al. (1988) J.Immunol. 140:3324-3330; P. S. Linsley et al. (1991) J. Exp. Med.173:721-730; C. D. Gimmi, et al. (1991) Proc. Natl. Acad. Sci. USA88:6575-6579; J. W. Young et al. (1992) J. Clin. Invest. 90:229-237; L.Koulova et al. (1991) J. Exp. Med. 173:759-762; H. Reiser et al. (1992)Proc. Natl. Acad. Sci. USA 89:271-275; G. A. van-Seventer et al. (1990)J. Immunol. 144:4579-4586; J. M. LaSalle et al. (1991) J. Immunol.147:774-80; M. I. Dustin et al. (1989) J. Exp. Med. 169:503; R. J.Armitage et al. (1992) Nature 357:80-82; Y. Liu et al. (1992) J. Exp.Med. 175:437-445). Considerable evidence suggests that B7, an APCcell-surface protein, is one such costimulatory molecule (P. S. Linsleyet al. (1991) J. Exp. Med. 173:721-730; C. D. Gimmi et al. (1991) Proc.Natl. Acad. Sci. USA 88:6575-6579; L. Koulova et al. (1991) J. Exp. Med.173:759-762; H. Reiser et al. (1992) Proc. Natl. Acad. Sci. USA 89,271-275; P. S. Linsley et al. (1990) Proc. Natl. Acad. Sci. USA87:5031-5035; G. J. Freeman et al. (1991) J. Exp. Med. 174:625-631).

B7 has been shown to bind to two counter-receptors (ligands) expressedon T-cells, termed CD28 and CTLA-4. B7 binding to CD28 induces T-cellsto proliferate and secrete IL-2 (P. S. Linsley et al. (1991) J. Exp.Med. 173, 721-730; C. D. Gimmi et al. (1991) Proc. Natl. Acad. Sci. USA88:6575-6579; C. B. Thompson et al. (1989) Proc. Natl. Acad. Sci. USA86:1333-1337; C. H. June et al. (1990) Immunol. Today 11:211-6; F. A.Harding et al. (1992) Nature 356:607-609), allowing full T-cellactivation. Conversely, B7 binding to CTLA-4 mediates T-celldown-regulation. The importance of the B7:CD28/CTLA-4 costimulatorypathway has been demonstrated in vitro and in several in vivo modelsystems. Blockade of this pathway results in the development of antigenspecific tolerance in murine and humans systems (F. A. Harding et al.(1992) Nature 356:607-609; D. J. Lenschow et al. (1992) Science257:789-792; L. A. Turka et al. (1992) Proc. Natl. Acad. Sci, USA89:11102-11105; C. D. Gimmi et al. (1993) Proc. Natl. Acad. Sci. USA90:6586-6590). Conversely, the ectopic expression of B7 in B7 negativemurine tumor cells induces T-cell mediated specific immunity accompaniedby tumor rejection and long lasting protection to tumor challenge (L.Chen et al. (1992) Cell 71:1093-1102; S. E. Townsend et al. (1993)Science 259:368-370; S. Baskar et al. (1993) Proc. Natl. Acad. Sci. USA90:5687-5690). Therefore, manipulation of the B7:CD28/CTLA-4 pathwayoffers great potential to stimulate or suppress immune responses inhumans.

In addition to the previously characterized B7 molecule (referred tohereafter as B7-1) B7-1-like molecules have been identified (see, e.g.,M. Azuma et al. (1993) Nature 366:76-79; C. Chen et al. (1994) J.Immunol. 152:4929-36; R. H. Reeves et al. (1997) Mamm. Genome 8:581-582;K. Ishikawa et al. (1998) DNA Res. 5:169-176; U.S. Pat. No. 5,942,607issued Aug. 24, 1999 to Freeman et al.). In particular, PD-L1 and PD-L2have been identified as inhibitors of T-cell activation (G. J. Freemanet al. (2000) J. Exp. Med. 192:1027-1034; Y. Latchman et al., (2001)Nature Immunology 2:261-268), whereas B7-H1, B7-H3, and B7-DC have beendescribed as co-stimulators of T-cell proliferation (H. Dong et al.(1999) Nature Medicine 5:1365-1369; A. I. Chapoval (2001) NatureImmunology 2:269-274; Tseng et al. (2001) J. Exp. Med. 193(7):839-45).

Thus, there is a growing family of factors related to B7-1, whichmodulate T-cell activation (reviewed by J. Henry et al. (1999) Immunol.Today 20:285-288). The identification, isolation, and characterizationof B7-related factors are therefore important goals for the furtherunderstanding of T-cell activation and function in both normal anddisease states in animals, particularly humans. Accordingly, the presentinvention discloses the discovery and characterization of threeB7-related factors, termed BSL1, BSL2, and BSL3. Also disclosed arevarious assays and treatments utilizing the BSL1, BSL2, and BSL3factors.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide isolated nucleicacids encoding B7-related polypeptides that modulate inflammatory andimmune responses, including T-cell activation. B7-related polypeptideswithin the scope of the invention include counter-receptors on thesurface of APCs capable of binding CD28 and/or CD28-related ligand(s).Specifically, B7-related polypeptides include the BSL1, BSL2, and BSL3polypeptides, and soluble fragments or derivatives thereof. Morespecifically, the B7-related nucleic acid is:

i) a nucleic acid molecule comprising at least a fragment of anucleotide sequence encoding a BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7,11, or 13), or BSL3 (SEQ ID NO:15) polypeptide;

ii) a nucleic acid molecule comprising a nucleotide sequence encoding apolypeptide that shares moderate to substantial sequence homology with aBSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ IDNO:15) polypeptide;

iii) a nucleic acid molecule capable of hybridizing to the BSL1 (SEQ IDNO:1 or 3), BSL2 (SEQ ID NO:6, 10, or 12), or BSL3 (SEQ ID NO:14)nucleotide sequences, or fragments thereof, under appropriate conditions(e.g., moderate or high stringency hybridization conditions);

iv) a nucleic acid molecule which differs from the nucleotide sequenceof BSL1 (SEQ ID NO:1 or 3), BSL2 (SEQ ID NO:6, 10, or 12), or BSL3 (SEQID NO:14) due to degeneracy in the genetic code, or recombinant orsynthetic modifications; or

v) a nucleic acid molecule that shares at least substantial homologywith the nucleic acid sequence set forth in SEQ ID NO:1, 3, 6, 10, 12,or 14.

In addition, nucleic acid probes useful for assaying a biological samplefor the presence of APCs expressing the BSL1, BSL2, and BSL3 factors areencompassed by the present invention.

It is another object of the present invention to provide vectors (e.g.,expression vectors) and fusion constructs comprising nucleic acidsencoding B7-related polypeptides. Expression vectors direct thesynthesis of the corresponding polypeptides or peptides in a variety ofhosts, particularly eukaryotic cells, such as mammalian and insect cellculture, and prokaryotic cells, such as Escherichia coli. Expressionvectors within the scope of the invention comprise a nucleic acidsequence encoding at least one B7-related polypeptide as describedherein, and a promoter operatively linked to the nucleic acid sequence.In one embodiment, the expression vector comprises a DNA sequenceencoding the extracellular domain of BSL1 (SEQ ID NO:2), BSL2 (SEQ IDNO:7, 11, or 13), or BSL3 (SEQ ID NO:15) fused to a DNA sequenceencoding the Fc region human immunoglobulin G1 (IgG1). Such expressionvectors can be used to transform or transfect host cells to therebyproduce polypeptides or peptides, including fusion proteins or peptidesencoded by nucleic acid molecules as described herein.

It is yet another object of the present invention to provide isolatedB7-related polypeptides, including the BSL1, BSL2, and BSL3polypeptides, or portions or derivatives thereof. Preferred B7-relatedpolypeptides comprise the amino acid sequences of the BSL1 (SEQ IDNO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15)polypeptides, or portions thereof. Such polypeptides comprise at least aportion of the mature forms of the BSL1, BSL2, and BSL3 polypeptides,and preferably comprise soluble forms of these polypeptides. Alsoencompassed by the present invention are polypeptides that sharemoderate to substantial homology with the amino acid sequence set forthin SEQ ID NO:2, 7, 11, 13, or 15, which are naturally occurring isoformsof the BSL1, BSL2, or BSL3 polypeptides, or modified recombinantpolypeptides.

It is still another object of the present invention to provide isolatedfusion proteins comprising the B7-related polypeptides, or portions orderivatives thereof, as disclosed herein. In one aspect, the fusionprotein comprises an extracellular domain portion of a B7-relatedpolypeptide fused to another polypeptide that alters the solubility,purification, binding affinity, and/or valency of the B7-relatedpolypeptide. Preferably, a DNA molecule encoding an extracellular domainportion of the BSL1, BSL2, or BSL3 polypeptides can be joined to DNAencoding the Fc region of human IgG1 to form DNA fusion products thatencode the BSL1-Ig, BSL2-Ig, or BSL3-Ig fusion proteins.

It is a further object of the present invention to provide methods ofisolating and identifying the corresponding counter-receptor(s) of theB7-related polypeptides, utilizing the isolated B7-related polypeptides,fusion proteins, or cognate antibodies disclosed herein. In oneembodiment, isolated BSL1, BSL2, or BSL3 polypeptides, or portionsthereof, can be incubated with protein extracts obtained from immune orinflammatory response cells, such as T-cells, to form a BSL/receptorcomplex, and then incubated with anti-BSL antibodies to isolate theBSL/receptor complex. Alternatively, a fusion protein comprising theBSL1, BSL2, or BSL3 polypeptide can be incubated with protein extractsobtained from immune or inflammatory response cells, such as T-cells,and then incubated with antibodies that specifically react with thefusion protein. Receptors that bind to the B7-related polypeptides wouldbe expected to have significant immunomodulatory activity.

It is another object of the present invention to provide diagnosticmethods and kits utilizing the B7-related factors of the presentinvention, including nucleic acids, polypeptides, antibodies, orfunctional fragments thereof. Such factors can be used, for example, indiagnostic methods and kits for measuring expression levels ofB7-related factors, and to screen for various B7-related diseases. Inaddition, the B7-related nucleic acids described herein can be used toidentify chromosomal abnormalities affecting BSL1, BSL2, or BSL3, and toidentify allelic variants or mutations of BSL1, BSL2, or BSL3 in anindividual or population.

It is yet another object of the present invention to provide isolatedantibodies, including monoclonal and polyclonal antibodies, that arespecifically reactive with the B7-related polypeptides, fusion proteins,or portions or derivatives thereof, as disclosed herein. Preferably,monoclonal antibodies are prepared to be specifically reactive with theBSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ IDNO:15) polypeptides, or portions or derivatives thereof.

It is another object of the present invention to provide methods ofimmunomodulation of a human or animal subject by the administration ofcompositions of the B7-related polypeptides, fusion proteins, orportions or derivatives thereof, as disclosed herein. Such compositionswould be expected to up-regulate or down-regulate the activities ofimmune or inflammatory response cells (e.g., T-cells). For example,B7-related polypeptides in a composition may interact with CD28 andthereby up-regulate immune cell activity. Alternatively, B7-relatedpolypeptides in a composition may interact with CTLA-4 and therebydown-regulate immune cell activity. In one embodiment, compositions ofBSL1-Ig, BSL2-Ig, and BSL3-Ig, fusion proteins are administered, e.g.via injection, to a subject to provide systemic immunosupression orimmunostimulation. Such compositions can be administered alone, or incombination with one or more immunomodulatory molecules.

It is still another object of the present invention to provide methodsof immunomodulation of a human or animal subject by the administrationof compositions of antibodies that are specifically reactive with theB7-related polypeptides, fusion proteins, or portions or derivativesthereof, as disclosed herein. Such compositions can be expected to blockthe co-stimulatory activities of the B7-related polypeptides, and todown-regulate immune or inflammatory response cells (e.g., T-cells),accordingly. In one embodiment, compositions of monoclonal antibodiesthat are specifically reactive with the BSL1 (SEQ ID NO:2), BSL2 (SEQ IDNO:7, 11, or 13), or BSL3 (SEQ ID NO:15) polypeptides, or fragmentsthereof, are administered, e.g., via injection, to a subject to provideimmunosupression or induced tolerance. Such compositions can beadministered alone, or in combination with one or more immunomodulatorymolecules. The methods of inducing tolerance described herein can beused prophylactically for preventing immune responses such astransplantation rejection (solid organ and bone marrow) and graft versushost disease, especially in autologous bone marrow transplantation. Suchmethods can also be useful therapeutically, in the treatment ofautoimmune diseases, transplantation rejection, and established graftversus host disease in a subject.

It is a further object of the present invention to provide methods ofthe immunomodulation of a human or animal subject by the administrationof compositions of genetically engineered vectors or cells comprisingthe B7-related polypeptide expression cassettes as disclosed herein. Ina preferred embodiment, the cells are antigen presenting cells, such asa macrophages, which are transfected or transduced to allow expressionof one or more of the B7-related polypeptides, including the BSL1 (SEQID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15)polypeptides, or fragments or derivatives thereof, and then introducede.g., via transplantation, into the recipient. Consistent with thepresent invention, the genes encoding the BSL1, BSL2, or BSL3polypeptides can be transfected or transduced alone, or in combinationwith genes encoding other immunomodulatory molecules.

Additional objects and advantages afforded by the present invention willbe apparent from the detailed description and exemplificationhereinbelow.

DESCRIPTION OF THE FIGURES

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects. In the figures of the present invention, thenucleotide and amino acid sequences are represented by their one-letterabbreviations.

FIGS. 1A-1C illustrate the nucleotide and predicted amino acid sequenceof BSL1. FIG. 1A shows the nucleotide sequence of BSL1 (SEQ ID NO:1)determined from the full-length clone isolated from a cDNA libraryprepared from human microvascular endothelial cells treated withTNF-alpha; nucleotides 1-92 contain the 5′-untranslated region;nucleotides 93-95 contain the translation initiation signal (ATG);nucleotides 93-962 encode the protein coding region; nucleotides 963-965contain the translation termination signal (TAA); nucleotides 963-1576contain the 3′-untranslated region; and nucleotides 1577-1605 containthe poly(A)⁺ RNA tail. FIG. 1B shows the predicted amino acid sequenceof BSL1 (SEQ ID NO:2); amino acids 1-240 contain the predictedextracellular domain (ECD). FIG. 1C shows the nucleotide sequence ofBSL1 (SEQ ID NO:3) determined from the full-length clone isolated from acDNA library prepared from GM-CSF/IL-4 differentiated human monocytecells; nucleotides 1-92 contain the 5′-untranslated region; nucleotides93-95 contain the translation initiation signal (ATG); nucleotides93-962 contain the protein coding sequence; nucleotides 963-965 containthe translation stop signal (TAA); and nucleotides 963-3621 contain the3′-untranslated region (unique sequence is shown in bold).

FIGS. 2A-2B illustrate the nucleotide and predicted amino acid sequenceof the BSL1-Ig fusion construct. FIG. 2A shows the nucleotide sequenceof the BSL1-Ig fusion construct (SEQ ID NO:4); nucleotides 1-72 encodethe predicted CD5 signal sequence; nucleotides 73-78 contain the SpeIrestriction site; nucleotides 78-729 encode the predicted BSL1 ECD;nucleotides 730-1440 encode the Fc portion of human IgG1; andnucleotides 1441-1443 contain the translation stop signal (TGA). FIG. 2Bshows the BSL1-Ig predicted amino acid sequence (SEQ ID NO:5).

FIGS. 3A-3F illustrate the nucleotide and predicted amino sequences ofthe BSL2 clones. FIG. 3A shows the nucleotide sequence of theBSL2-4616811 clone (SEQ ID NO:6); nucleotides 1-12 include vectorsequence; nucleotides 121-123 contain the translation initiation signal(ATG); between nucleotides 204-205 is the predicted signal peptidecleavage site; nucleotides 1516-1587 encode the predicted transmembranedomain; nucleotides 1723-1725 contain the translation termination signal(TGA). FIG. 3B shows the predicted amino acid sequence of theBSL2-4616811 clone (SEQ ID NO:7); amino acids (1-465) contain thepredicted ECD. FIG. 3C shows the nucleotide sequence of the BSL2-L165-21clone (SEQ ID NO:10); nucleotides 1-3 encode the translation initiationsignal (ATG); between nucleotides 84-85 is the predicted signal peptidecleavage site; nucleotides 742-813 encode the predicted transmembranedomain; nucleotides 949-951 contain the translation termination signal(TGA). FIG. 3D shows the predicted amino acid sequence of theBSL2-L165-21 clone (SEQ ID NO:11); amino acids 1-247 contain thepredicted ECD. FIG. 3E shows the nucleotide sequence of theBSL2-L165-35b clone (SEQ ID NO:12); nucleotides 1-3 encode thetranslation initiation signal (ATG); between nucleotides 84-85 is thepredicted signal peptide cleavage site; nucleotides 742-813 encode thepredicted transmembrane domain; nucleotides 949-951 contain thetranslation termination signal (TGA). FIG. 3F shows the predicted aminoacid sequence of the BSL2-L165-35b clone (SEQ ID NO:13); amino acids1-247 contain the predicted ECD.

FIGS. 4A-4B illustrate the nucleotide and predicted amino sequences ofthe of the BSL2-4616811-Ig fusion construct. FIG. 4A shows thenucleotide sequence of the BSL2-4616811-Ig clone (SEQ ID NO:8):nucleotides 1-3 contain the translation initiation signal (ATG);nucleotides 1-1394 encode the native BSL2-4616811 sequence; nucleotide1395 is a silent mutation introduced to facilitate construction of thefusion protein; nucleotides 1396-2097 encode the Fc portion of humanIgG1; nucleotides 2095-2097 contain the translation termination signal(TGA). FIG. 4B shows the predicted amino acid sequence of theBSL2-4616811-Ig fusion protein (SEQ ID NO:9); amino acids 1-465 ofcontain the native sequence of BSL2-4616811; amino acids 466-698 containthe Fc domain of human IgG.

FIGS. 5A-5B illustrate the nucleotide and predicted amino acid sequenceof BSL3. FIG. 5A shows the nucleotide sequence of BSL3 (SEQ ID NO:14):nucleotides 1-326 contain 5′ untranslated region; nucleotides 327-329contain the translation initiation signal (ATG); nucleotides 981-1055encode a predicted transmembrane domain; nucleotides 1146-1148 containthe translation termination signal (TGA) FIG. 5B shows the BSL3predicted amino acid sequence (amino acids 1-273) (SEQ ID NO:15); aminoacids 1-219 contain the predicted ECD.

FIGS. 6A-6B illustrate the nucleotide and predicted amino sequences ofthe of the BSL3-Ig fusion construct. FIG. 6A shows the nucleotidesequence of BSL3-Ig (L232-6) (SEQ ID NO:16): nucleotides 1-3 contain thetranslation initiation signal (ATG); nucleotides 1-651 encode the nativeBSL3 sequence; nucleotides 652-654 encode an artificial sequenceintroduced during construction; nucleotides 655-1356 encode the Fcdomain of human IgG. FIG. 6B shows the predicted amino acid sequence ofBSL3-Ig (L232-6) (SEQ ID NO:17); amino acids 1-217 contain the nativeBSL3 sequence; amino acid 218 contains an artificial sequence introducedduring construction; amino acids 219-451 contain the Fc domain of humanIgG.

FIGS. 7A-7H illustrate the reagents and results of expression analysisperformed for BSL1, BSL2, and BSL3. FIG. 7A shows the nucleotidesequence of the BSL1 probe (SEQ ID NO:18) used for northern blotanalysis. FIG. 7B shows the nucleotide sequence of the BSL2 probe (SEQID NO:19) used for northern blot analysis. FIG. 7C shows the nucleotidesequence of the BSL3 probe (SEQ ID NO:20). FIG. 7D shows the levels ofBSL1, BSL3, and BSL3 mRNA observed in various cell types as determinedby northern blot analysis; “PBT” indicates peripheral blood T-cells;“CD3/CD28” indicates stimulation with anti-CD3 and anti-CD28 antibodies;“PMA” indicates stimulation with phorbol 12 myristate 13 acetate; “LPS”indicates stimulation with lipopolysaccharide; “PBM” indicatesperipheral blood monocytes; “PHA” indicates stimulation withphytohemaglutinin; “GM-CSF/IL-4” indicates stimulation with GM-CSF andIL-4; “HMVEC” indicates human microvascular endothelial cells;“TNF-alpha” indicates stimulation with TNF-alpha; and “H292 (Starved)indicates serum starved H292 cells. FIG. 7E shows BSL3 expression levelsin various tissue types as determined by northern analysis ofcommercially available blots using radiolabeled BSL3/KpnI+XbaI probe.FIG. 7F shows BSL3 expression levels in various tissue types asdetermined by hybridization analysis of commercially availablemicroarrays using radiolabeled BSL3/KpnI+XbaI probe. FIG. 7G shows BSL1expression levels in various tissue types as determined by quantitativePCR. FIG. 7H shows BSL3 expression levels in various tissue types asdetermined by quantitative PCR.

FIGS. 8A-8E illustrate the results of PCR analysis performed todetermine the relative levels of the BSL2-4616811 or BSL2-L165-35btranscripts in various cell types, with or without stimulation. The toparrow points to the bands representing the BSL2-4616811 transcript; thebottom arrow points to the bands representing the BSL2-L165-35btranscript. FIG. 8A shows the results for Raji, Ramos, PM-LCL, andPL-LCL cell types, with or without PMA and ionomycin stimulation. FIG.8B shows the results for CE-LCL cells, HL60, Thp1, and HUVEC cell types,with or without stimulation. FIG. 8C shows the results for peripheralblood T-cells with or without PMA and ionomycin stimulation. The resultsfrom cells isolated from two separate donors are shown (donor 079 anddonor 124). FIG. 8D shows the results for CEM and HUT78 cells, with orwithout PMA and ionomycin stimulation. FIG. 8E shows the results of aPCR reaction using BSL2-4616811 plasmid as template. Lane 1: LambdaBstEII DNA ladder; lane 2: PCR product. The upper arrow points to thesize of the predicted PCR product from the BSL2-4616811 transcript. Thelower arrow points to the size of the predicted PCR product for theBSL2-L165-35b transcript. The results demonstrate that the forwardprimer preferentially binds the specific binding site in the firstvariable fold rather than for the homologous site in the second variablefold of BSL2-4616811.

FIGS. 9A-9F illustrate the results of fluorescence activated cellsorting (FACS) performed using BSL1, BSL2, and BSL3 monoclonalantibodies. FIG. 9A shows FACS analysis of A549 epithelial lung cellsusing BSL1 monoclonal antibodies. Column 1: no monoclonal antibodies;column 2: isotype control; column 3: BSL1 hybridoma supernatant 32. FIG.9B shows FACS analysis of A549 epithelial lung cells using BSL2-4616811monoclonal antibodies. Column 1: no monoclonal antibodies; column 2:isotype control; column 3: BSL2 MAb 1F7G2; column 4: BSL2 Mab 2B10D7;column 5: BSL2 Mab 3E6D3; column 6: BSL2 Mab 4C2C6; column 7: B5L2 Mab5D7E2. FIG. 9C shows FACS analysis of various cell types using BSL3monoclonal antibodies. FIG. 9D shows FACS analysis of human umbilicalvein endothelial cells (HUVEC) with or without TNF-alpha stimulationusing BSL3 monoclonal antibodies. FIGS. 9E-9F shows FACS analysis ofperipheral blood monocytes (PBMCs) with or without GM-CSF/1L4 or PHAstimulation using BSL3 monoclonal antibodies. FIG. 9E shows results fromcells isolated from donor 126; FIG. 9F shows results from cells isolatedfrom donor 145.

FIGS. 10A-10E illustrate co-stimulation of peripheral blood T-cellsusing BSL2-4616811-Ig (BSL2vcvclg) and BSL3-Ig fusion proteins in thepresence of CD3 monoclonal antibody. The L6-Ig fusion protein is used asa negative control. FIG. 10A shows results from cells isolated fromdonor 010. FIG. 10B shows results from cells isolated from donor 127.FIGS. 10C-10D show results from cells isolated from donor 78. FIGS.10E-10F show results from cells isolated from donor 124. FIGS. 10G-10Jillustrate the blockade of co-stimulation of peripheral blood T-cellsusing BSL2 or BSL3 monoclonal antibodies. FIGS. 10G and 10I show theresults from cells stimulated with CD3 monoclonal antibodies andBSL2-4616811-Ig (BSL2vcvclg) and blockaded with BSL2 monoclonalantibodies. Column 1: BSL2-1F7G2 MAb; column 2: BSL2-2B10D7 MAb; column3: BSL2-3E6D3 MAb; column 4: BSL2-5D7E2 MAb; and column 5: isotypecontrol antibody. FIG. 10G shows results from cells isolated from donor010; FIG. 10I shows results from cells isolated from donor 127. FIGS.10H and 10J show the results from cells stimulated with CD3 monoclonalantibodies and BSL3-Ig fusion protein and blockaded with BSL3 monoclonalantibodies. Column 1: BSL3-1A4A1 MAb; column 2: BSL3-2B6H7 MAb; andcolumn 3: isotype control antibody. FIG. 10H shows results from cellsisolated from donor 010; FIG. 10J shows results from cells isolated fromdonor 127.

DETAILED DESCRIPTION OF THE INVENTION

Identification of B7-Related Factors

In accordance with the methods of the present invention, threeB7-related factors, designated BSL1, BSL2, and BSL3, have beenidentified and characterized. Such B7-related factors may provide amolecular basis for the activation of immune or inflammatory responsecells, such as T-cells, at different times and in different illnessesand disease states. In addition, the disclosed B7-related factors can beutilized in the prevention or treatment certain diseases by modulatingthe activity of immune or inflammatory response cells, such as T-cells,using the methods described in detail herein. Such methods can be usedas prophylaxis or treatments for cancers or immune-related disorders asdetailed below.

Identification of B7-Related Genes from cDNA Libraries:

To identify B7-related factors, cDNA libraries can be constructed andanalyzed using several well-established techniques. Messenger RNA can beobtained from cells expressing B7-1 and/or B7-related factors. Forexample, mRNA can be obtained from differentiated human peripheral bloodmononuclear cells. Alternatively, mRNA can be obtained from varioussubsets of neoplastic B cells, including tumor cells isolated frompatients with non-Hodgkin's lymphoma (L. Chaperot et al. (1999) Exp.Hematol. 27:479-88). Such cells are known to express B7-1 and, thus, mayexpress B7-related factors, and can also serve as a source of the mRNAfor construction of the cDNA library.

Total cellular mRNA can be isolated by a variety of techniques, e.g.,guanidinium-thiocyanate extraction (J. M. Chirgwin et al. (1979)Biochemistry 18:5294-5299; Chomczynski et al. (1987) Anal. Biochem.162:156-9). Following isolation, poly(A)⁺ RNA can be purified usingoligo(dT) cellulose. The purified poly(A)⁺ RNA can then be used as atemplate for cDNA synthesis utilizing reverse transcriptase polymerasechain reaction (RT-PCR; see C. R. Newton et al. (1997) PCR 2^(nd) Ed,Scientific Publishers, Oxford, England). Following reversetranscription, the cDNA can be converted to double stranded DNA usingconventional techniques (see H. Okayama et al. (1982) Mol. Cell. Biol.2:161; U. Gubler et al. (1983) Gene 25:263).

Cloning of the double stranded cDNAs can be accomplished usingtechniques that are well known in the art (see J. Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y.; F. M. Ausubel et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.). The use ofsynthetic adaptors prior to cloning is particularly preferred, since itobviates the need for cleavage of the cDNA with one or more restrictionenzymes (see, for example, E. C. Bottger (1989) Biotechniques 7:925-6,928-90). Using this method, non-self complementary, kinased adaptors canbe added to the DNA prior to ligation with the vector. Virtually anyadaptor can be employed.

A cDNA library sequence can be expressed when placed in the senseorientation in a vector that supplies an appropriate promoter. Vectorsmay also include an origin of replication and various enhancersequences, splice acceptor/donor sequences, and polyadenylationsequences. Vectors may further include a marker that allows forselection of cells containing the vector construct. Markers may be aninducible or non-inducible gene and will generally allow for positiveselection under induction, or without induction, respectively. Examplesof marker genes include neomycin, dihydrofolate reductase, glutaminesynthetase, and the like. Notably, prepared cDNA libraries can beobtained from various commercial sources (e.g., Incyte Genomics, Inc.,St. Louis, Mo.; Stratagene, La Jolla, Calif.)

The cDNA library can be used to clone B7-related factors utilizingexpression cloning techniques (see B. Seed et al. (1987) Proc. Natl.Acad. Sci, USA 84:3365-3369; A. Aruffo et al. (1987) Proc. Natl. Acad.Sci. USA 84:8573-8577). In one embodiment, plasmid DNA is introducedinto a cell line by known methods of transfection (e.g., DEAE-Dextran)and allowed to replicate and express the cDNA inserts. B7-1 antigen isdepleted from the transfected cells using an anti-B7-1 monoclonalantibody (e.g., 133 and B1.1) and anti-murine IgG and IgM coatedimmunomagnetic beads. Transfectants expressing B7-related factors arepositively selected by incubation with CTLA-4-Ig and CD28-Ig followed bypanning with anti-human Ig immunoglobulin. After panning, episomal DNAis recovered from the panned cells and transfected into a competentbacterial host, preferably Escherichia coli (E. coli). Plasmid DNA issubsequently reintroduced into the cell line and the cycle of expressionand panning repeated at least two times. Following the final panningcycle, plasmid DNA is prepared from individual colonies, transfectedinto the cell line and analyzed for expression of the B7-relatedpolypeptides by indirect immunofluorescence with CTLA-4-Ig and CD28-Ig.After cloning, plasmids are prepared from the clones strongly reactivewith the CTLA-4-1 g, and then sequenced using conventional sequencingtechniques (reviewed in G. W. Slater et al. (1998) Electrophoresis19:1525-41).

Identification of B7-Related Genes in Protein Sequence Databases:

Alternatively, B7-related factors can be identified by screeningavailable sequence databases. The polypeptide sequence encoded by apreviously identified B7 factor (e.g., B7-1, B7-2, or B7-H1) or aB7-related factor disclosed herein (e.g., BSL1, BSL2, or BSL3), can becompared with the polypeptide sequences present in various proteindatabases. Publicly available protein sequence databases, e.g., GENBANK®GenPept, SWISS-PROT®, Protein Data Bank (PDB), Protein InformationResource (PIR), Human UniGene (National Center for BiotechnologyInformation), can be used to determine if additional B7-related factorsare present in mammalian, preferably human, species. Alternatively,privately owned protein sequence databases, e.g., the Incyte Genomicssequence database (Incyte Genomics), can be used to identify B7-relatedfactors. Databases with relatively few redundant sequences, e.g., PIR orSWISS-PROT® databases, can be used to improve the statisticalsignificance of a sequence match. However, databases which are morecomprehensive and up-to-date, e.g., GENBANK®, GenPept, and IncyteGenomics sequence databases (Incyte Genomics), are preferred.

Any method known in the art can be used to align and compare thepreviously identified B7 factor sequence with the sequences present inthe protein sequence databases. Preferably, the BLAST program is used(S. F. Altschul et al. (1990) J. Mol. Biol. 215:403-410; S. Karlin etal. (1990) Proc. Natl. Acad. Sci. USA 87:2264-68; S. Karlin et al.(1993) Proc. Natl. Acad. Sci. USA 90:5873-7). BLAST identifies localalignments between the sequence of the previously identified protein andthe protein sequences in the database, and predicts the probability ofthe local alignment occurring by chance. Although the original BLASTprograms utilized ungapped local alignments, more recently developedBLAST programs such as WU-BLAST2/BLAST v2.0 (S. F. Altschul et al.(1996) Methods Enzymol. 266, 460-480) have been modified to incorporategapped local alignments similar to SSEARCH (T. F. Smith et al. (1981) J.Mol. Biol. 147:195-197) and FASTA programs (W. R. Pearson (1990) MethodsEnzymol. 183:63-98). In addition, position-specific-iterated BLAST(PSI-BLAST) programs have been developed to identify weak butbiologically relevant sequence similarities (S. F. Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402). Furthermore,pattern-hit-initiated BLAST (PHI-BLAST) programs have been designed toidentify specific patterns or sequence motifs shared bydistantly-related proteins (Z. Zhang et al. (1998) Nucleic Acids Res.26:3986-3990). Specialized BLAST programs are also available forperforming searches of human, microbial, and malaria genome sequences,as well as searches for vector, immunoglobulin, and predicted humanconsensus sequences (National Center for Biotechnology Information(NCBI), Bethesda, Md.).

Both FASTA and BLAST programs identify very short exact sequence matchesbetween the query sequence and the databases sequences, analyze the bestshort sequence matches (“hits”) to determine if longer stretches ofsequence similarity are present, and then optimize the best hits bydynamic programming (S. F. Altschul et al. (1990) J. Mol. Biol.215:403-410; W. R. Pearson, supra). In contrast, the SSEARCH programcompares the query sequence to all the sequences in the database viapair-wise sequence comparisons (T. F. Smith et al., supra). Thus, theSSEARCH program is considered more sensitive than the BLAST and FASTAprograms, but it is also significantly slower. The BLAST and FASTAprograms utilize several approximations to increase their searchingspeed, and utilize statistical parameters (see below) to increasesensitivity and selectivity to approximate the performance of theSSEARCH program. A particular sequence alignment program can be chosenbased on the requirements of a sequence search, or individualpreferences. In some cases, it may be necessary to use more than onesearch alignment program to confirm search alignment results or resolveambiguous search results.

Typically, BLAST analysis employs (i) a scoring matrix (such as, e.g.,BLOSSUM 62 or PAM 120) to assign a weighted homology value to eachresidue and (ii) a filtering program(s) (such as SEG or XNU) thatrecognizes and eliminates highly repeated sequences from thecalculation. An appropriate homology cutoff is then determined byperforming BLAST comparisons (using a particular scoring matrix andfiltering program) between sequences that are known to be related. Itwill be understood that other appropriate scoring matrices and filteringprograms may be used when the cutoff is calibrated as described herein.That is, the particular cutoff point may vary when different standardparameters are used, but it will correspond to the P(N) scores exhibitedwhen highly related sequences are compared using those particularparameters.

B7-Related Nucleic Acids

One aspect of the present invention pertains to isolated nucleic acidshaving a nucleotide sequence such as BSL1 (SEQ ID NO:1 or 3), BSL2 (SEQID NO:6, 10, or 12), or BSL3 (SEQ ID NO:14), or fragments thereof. Thenucleic acid molecules of the invention can be DNA or RNA. A preferrednucleic acid is a DNA encoding the human BSL1 (SEQ ID NO:2), BSL2 (SEQID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15), or fragments or functionalequivalents thereof. Such nucleic acids can comprise at least 15, 20,25, 50, 60, 100, 200, 240, 255, 270, 300, 305, 310, 410, 500, 630, 700,or 1000 contiguous nucleotides.

The term “isolated” as used throughout this application refers to aB7-related nucleic acid, polypeptide, peptide, protein fusion, orantibody, that is substantially free of cellular material or culturemedium. An isolated or substantially purified molecule contains lessthan about 50%, preferably less than about 25%, and most preferably lessthan about 10%, of the cellular components with which it was associated.

The term “functional equivalent” is intended to include nucleotidesequences encoding functionally equivalent B7-related factors. Afunctional equivalent of a B7-related protein includes fragments orvariants that perform at least one characteristic function of theB7-related protein (e.g., ligand-binding, antigenic, intra-, orintercellular activity). For example, DNA sequence polymorphisms withinthe nucleotide sequence of a B7-related factor, especially those withinthe third base of a codon, may result in “silent” mutations, which donot affect the encoded amino acid sequence of the protein due to thedegeneracy of the genetic code.

Preferred embodiments include an isolated nucleic acid sharing at least60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5, or 100% sequence identity witha polynucleotide sequence of BSL1 (SEQ ID NO:1 or 3), BSL2 (SEQ ID NO:6,10, or 12), or BSL3 (SEQ ID NO:15). This polynucleotide sequence may beidentical to the nucleotide sequences of BSL1 (SEQ ID NO:1 and 3), BSL2(SEQ ID NO:6, 10, or 12), or BSL3 (SEQ ID NO:14), or may include up to acertain integer number of nucleotide alterations as compared to thereference sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing. Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073(1988).

For nucleic acids, sequence identity can be determined by comparing aquery sequences to sequences in publicly available sequence databases(NCBI) using the BLASTN2 algorithm (S. F. Altschul et al., 1997, Nucl.Acids Res., 25:3389-3402). The parameters for a typical search are:E=0.05, v=50, B=50, wherein E is the expected probability score cutoff,V is the number of database entries returned in the reporting of theresults, and B is the number of sequence alignments returned in thereporting of the results (S. F. Altschul et al., 1990, J. Mol. Biol.,215:403-410).

In another approach, nucleotide sequence identity can be calculatedusing the following equation: % identity=(number of identicalnucleotides)/(alignment length in nucleotides)*100. For thiscalculation, alignment length includes internal gaps but not terminalgaps. Alternatively, nucleotide sequence identity can be determinedexperimentally using the specific hybridization conditions describedbelow.

In accordance with the present invention, nucleic acid alterations areselected from the group consisting of at least one nucleotide deletion,substitution, including transition and transversion, insertion, ormodification (e.g., via RNA or DNA analogs, dephosphorylation,methylation, or labeling). Alterations may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Alterations of a nucleic acidsequence of BSL1 (SEQ ID NO:1 and 3), BSL2 (SEQ ID NO:6, 10, or 12), orBSL3 (SEQ ID NO:14) may create nonsense, missense, or frameshiftmutations in the coding sequence, and thereby alter the polypeptideencoded by the nucleic acid.

Also encompassed by the present invention are splice variants derivedfrom the BSL1 (SEQ ID NO:1 and 3), BSL2 (SEQ ID NO:6, 10, or 12), orBSL3 (SEQ ID NO:14) nucleic acid sequences. As used herein, the term“splice variant” refers to variant B7-related nucleic acids andpolypeptides produced by differential processing of the primarytranscript(s) of genomic DNA. An alternate splice variant may comprise,for example, any one of the sequences of BSL2 (SEQ ID NO:6, 10, or 12)disclosed herein. Alternate splice variants can also comprise othercombinations of introns/exons of BSL1, BSL2, or BSL3, which can bedetermined by those of skill in the art. Alternate splice variants canbe determined experimentally, for example, by isolating and analyzingcellular RNAs (e.g., Southern blotting or PCR), or by screening cDNAlibraries using the B7-related nucleic acid probes or primers describedherein. In another approach, alternate splice variants can be predictedusing various methods, computer programs, or computer systems availableto practitioners in the field.

General methods for splice site prediction can be found in Nakata, 1985,Nucleic Acids Res. 13:5327-5340. In addition, splice sites can bepredicted using, for example, the GRAIL™ (E. C. Uberbacher and R. J.Mural, 1991, Proc. Natl. Acad. Sci. USA, 88:11261-11265; E. C.Uberbacher, 1995, Trends Biotech., 13:497-500;http://grail.lsd.ornl.gov/grailexp); GenView (L. Milanesi et al., 1993,Proceedings of the Second International Conference on Bioinformatics,Supercomputing, and Complex Genome Analysis, H. A. Lim et al. (eds),World Scientific Publishing, Singapore, pp. 573-5881); SpliceView; andHSPL (V. V. Solovyev et al., 1994, Nucleic Acids Res. 22:5156-5163; V.V. Solovyev et al., 1994, “The Prediction of Human Exons byOligonucleotide Composition and Discriminant Analysis of Spliceable OpenReading Frames,” R. Altman et al. (eds), The Second Internationalconference on Intelligent systems for Molecular Biology, AAAI Press,Menlo Park, Calif., pp. 354-362; V. V. Solovyev et al., 1993,“Identification Of Human Gene Functional Regions Based OnOligonucleotide Composition,” L. Hunter et al. (eds), In Proceedings ofFirst International conference on Intelligent System for MolecularBiology, Bethesda, pp. 371-379) computer systems.

Additionally, computer programs such as GeneParser (E. E. Snyder and G.D. Stormo, 1995, J. Mol. Biol. 248: 1-18; E. E. Snyder and G. D. Stormo,1993, Nucl. Acids Res. 21(3): 607-613;http://mcdb.colorado.edu/˜eesnyder/GeneParser.html); MZEF (M. Q. Zhang,1997, Proc. Natl. Acad. Sci. USA, 94:565-568;http://argon.cshl.org/genefinder); MORGAN (S. Salzberg et al., 1998, J.Comp. Biol. 5:667-680; S. Salzberg et al. (eds), 1998, ComputationalMethods in Molecular Biology, Elsevier Science, New York, N.Y., pp.187-203); VEIL (J. Henderson et al., 1997, J. Comp. Biol. 4:127-141);GeneScan (S. Tiwari et al., 1997, CABIOS (BioInformatics) 13: 263-270);GeneBuilder (L. Milanesi et al., 1999, Bioinformatics 15:612-621);Eukaryotic GeneMark Besemer et al., 1999, Nucl. Acids Res.27:3911-3920); and FEXH (V. V. Solovyev et al., 1994, Nucleic Acids Res.22:5156-5163). In addition, splice sites (i.e., former or potentialsplice sites) in cDNA sequences can be predicted using, for example, theRNASPL (V. V. Solovyev et al., 1994, Nucleic Acids Res. 22:5156-5163);or INTRON (A. Globek et al., 1991, INTRON version 1.1 manual, Laboratoryof Biochemical Genetics, NIMH, Washington, D.C.) programs.

The present invention also encompasses naturally-occurring polymorphismsof BSL1 (SEQ ID NO:1 and 3), BSL2 (SEQ ID NO:6, 10, or 12), or BSL3 (SEQID NO:14). As will be understood by those in the art, the genomes of allorganisms undergo spontaneous mutation in the course of their continuingevolution generating variant forms of gene sequences (Gusella, 1986,Ann. Rev. Biochem. 55:831-854). Restriction fragment lengthpolymorphisms (RFLPs) include variations in DNA sequences that alter thelength of a restriction fragment in the sequence (Botstein et al., 1980,Am. J. Hum. Genet. 32, 314-331 (1980). RFLPs have been widely used inhuman and animal genetic analyses (see WO 90/13668; WO 90/11369;Donis-Keller, 1987, Cell 51:319-337; Lander et al., 1989, Genetics 121:85-99). Short tandem repeats (STRs) include tandem di-, tri- andtetranucleotide repeated motifs, also termed variable number tandemrepeat (VNTR) polymorphisms. VNTRs have been used in identity andpaternity analysis (U.S. Pat. No. 5,075,217; Armour et al., 1992, FEBSLett. 307:113-115; Horn et al., WO 91/14003; Jeffreys, EP 370,719), andin a large number of genetic mapping studies.

Single nucleotide polymorphisms (SNPs) are far more frequent than RFLPS,STRs, and VNTRs. SNPs may occur in protein coding (e.g., exon), ornon-coding (e.g., intron, 5′UTR, 3′UTR) sequences. SNPs in proteincoding regions may comprise silent mutations that do not alter the aminoacid sequence of a protein. Alternatively, SNPs in protein codingregions may produce conservative or non-conservative amino acid changes,described in detail below. In some cases, SNPs may give rise to theexpression of a defective or other variant protein and, potentially, agenetic disease. SNPs within protein-coding sequences can give rise togenetic diseases, for example, in the β-globin (sickle cell anemia) andCFTR (cystic fibrosis) genes. In non-coding sequences, SNPs may alsoresult in defective protein expression (e.g., as a result of defectivesplicing). Other single nucleotide polymorphisms have no phenotypiceffects.

Single nucleotide polymorphisms can be used in the same manner as RFLPsand VNTRs, but offer several advantages. Single nucleotide polymorphismstend to occur with greater frequency and are typically spaced moreuniformly throughout the genome than other polymorphisms. Also,different SNPs are often easier to distinguish than other types ofpolymorphisms (e.g., by use of assays employing allele-specifichybridization probes or primers). In one embodiment of the presentinvention, a BSL1, BSL2, or BSL3 nucleic acid contains at least one SNP.Various combinations of these SNPs are also encompassed by theinvention. In a preferred aspect, a B7-related SNP is associated with aimmune system disorder, such as the disorders described in detailherein.

Further encompassed by the present invention are nucleic acid moleculesthat share moderate homology with the BSL1 (SEQ ID NO:1 and 3), BSL2(SEQ ID NO:6, 10, or 12), or BSL3 (SEQ ID NO:14) nucleic acid sequences,and hybridize to the BSL1, BSL2, or BSL3 nucleic acid molecules undermoderate stringency hybridization conditions. More preferred are nucleicacid molecules that share substantial homology with the BSL1, BSL2, orBSL3 nucleic acid sequences and hybridize to the BSL1, BSL2, or BSL3nucleic acid molecules under high stringency hybridization conditions.As used herein, the phrase “moderate homology” refers to sequences whichshare at least 60% sequence identity with a reference sequence (e.g.,BSL1, BSL2 or BSL3), whereas the phrase “substantial homology” refers tosequences that share at least 90% sequence identity with a referencesequence. It is recognized, however, that polypeptides and the nucleicacids encoding such polypeptides containing less than theabove-described level of homology arising as splice variants or that aremodified by conservative amino acid substitutions (or substitution ofdegenerate codons) are contemplated to be within the scope of thepresent invention.

The phrase “hybridization conditions” is used herein to refer toconditions under which a double-stranded nucleic acid hybrid is formedfrom two single nucleic acid strands, and remains stable. As known tothose of skill in the art, the stability of the hybrid sequence isreflected in the melting temperature (T_(m)) of the hybrid (see F. M.Ausubel et al., Eds, (1995) Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., New York, N.Y.). The T_(m) decreases approximately0.5° C. to 1.5° C. with every 1% decrease in sequence homology. Ingeneral, the stability of a hybrid sequence is a function of the lengthand guanine/cytosine content of the hybrid, the sodium ionconcentration, and the incubation temperature. Typically, thehybridization reaction is initially performed under conditions of lowstringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

In accordance with the present invention, “high stringency” conditionscan be provided, for example, by hybridization in 50% formamide,5×Denhardt's solution, 5×SSPE, and 0.2% SDS at 42° C., followed bywashing in 0.1×SSPE and 0.1% SDS at 65° C. By comparison, “moderatestringency” can be provided, for example, by hybridization in 50%formamide, 5×Denhardt's solution, 5×SSPE, and 0.2% SDS at 42° C.,followed by washing in 0.2×SSPE and 0.2% SDS at 65° C. In addition, “lowstringency” conditions can be provided, for example, by hybridization in10% formamide, 5×Denhardt's solution, 6×SSPE, and 0.2% SDS at 42° C.,followed by washing in 1×SSPE and 0.2% SDS at 50° C. It is understoodthat these conditions may be varied using a variety of buffers andtemperatures well known to those skilled in the art.

In a preferred embodiment of the present invention, the nucleic acid isa DNA molecule encoding at least a portion of the B7-related factor. Anucleic acid molecule encoding a novel B7-related factor can be obtainedfrom mRNA present in activated B lymphocytes. It may also be possible toobtain nucleic acid molecules encoding B7-related factors from B cellgenomic DNA. Thus, a nucleic acid encoding a B7-related factor can becloned from either a cDNA or a genomic library in accordance with theprotocols described in detail herein. Nucleic acids encoding novelB7-related factors can also be cloned from genomic DNA or cDNA usingestablished polymerase chain reaction (PCR) techniques (see K. Mullis etal. (1986) Cold Spring Harbor Symp. Quant, Biol. 51:260; K. H. Roux(1995) PCR Methods Appl. 4:S185) in accordance with the nucleic acidsequence information provided herein. The nucleic acid molecules of theinvention, or fragments thereof, can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (see, for example, U.S. Pat. No. 4,598,049 toItakura et al.; U.S. Pat. No. 4,458,066 to Caruthers et al.; U.S. Pat.Nos. 4,401,796 and 4,373,071 to Itakura).

It will be appreciated by one skilled in the art that variations in oneor more nucleotides (up to about 3-4% of the nucleotides) of the nucleicacid molecules encoding novel B7-related factors may exist amongindividuals within a population due to natural allelic variation. Anyand all such nucleotide variations and resulting amino acidpolymorphisms are within the scope of the invention. Furthermore, theremay be one or more isoforms or related, cross-reacting family members ofthe B7-related factors described herein. Such isoforms or family membersare defined as polypeptides that are related in function and amino acidsequence to a B7-related factor (e.g., BSL1, BSL2, or BSL3), but encodedby genes at different loci. In addition, it is possible to modify theDNA sequence of B7-related factors using genetic techniques to produceproteins or peptides with altered amino acid sequences.

DNA sequence mutations can be introduced into a nucleic acid encoding aB7-related factor by any one of a number of methods, including those forproducing simple deletions or insertions, systematic deletions,insertions or substitutions of clusters of bases or substitutions ofsingle bases, to generate desired variants. Mutations of the B7-relatednucleic acid molecule to generate amino acid substitutions or deletionsare preferably obtained by site-directed mutagenesis. Site directedmutagenesis systems are well known in the art, and can be obtained fromcommercial sources (see, for example, Amersham Pharmacia Biotech, Inc.,Piscataway, N.J.). Guidance in determining which amino acid residues maybe substituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software (DNASTAR, Inc., Madison,Wis.). Mutant forms of the BSL1, BSL2, or BSL3 nucleic acid moleculesare considered within the scope of the present invention, where theexpressed polypeptide or peptide is capable modulating the activity ofimmune or inflammatory cells (e.g., T-cells).

A fragment of the nucleic acid molecule encoding a novel B7-relatedfactor is defined as a nucleotide sequence having fewer nucleotides thanthe nucleotide sequence encoding the entire amino acid sequence of theB7-related factor. Nucleic acid fragments which encode polypeptideswhich retain the ability to bind to their natural ligand(s) on immune orinflammatory response cells, such as T-cells, and either amplify orblock immune responses (as evidenced by, for example, lymphokineproduction and/or T-cell proliferation by T-cells that have received aprimary activation signal) are considered within the scope of theinvention. For example, nucleic acid fragments that encode polypeptidesor peptides of a B7-related factor that retain the ability of thepolypeptides or peptides to bind CD28 and/or CD28-related ligand(s) anddeliver a co-stimulatory signal to T-cells are within the scope of theinvention. Generally, the nucleic acid molecule encoding a fragment of aB7-related factor will be selected from the coding sequence for themature protein. However, in some instances it may be desirable to selectall or part of a fragment or fragments from the coding region thatincludes the leader sequence.

In one embodiment of the present invention, a nucleic acid moleculecorresponding to a fragment of a BSL1, BSL2, or BSL3 nucleic acidsequence can be used as a probe for assaying a biological sample for theexpression of one or more B7-related factors, or as a primer for DNAsequencing or PCR amplification. Preferably, such fragments are at least8 contiguous nucleotides in length, more preferably at least 12contiguous nucleotides in length, even more preferably at least 15contiguous nucleotides in length, and even more preferably at least 20contiguous nucleotides in length. Nucleic acid molecules within thescope of the invention may also contain linker sequences, modifiedrestriction endonuclease sites, and other sequences useful for molecularcloning, expression, or purification of recombinant protein or fragmentsthereof. Nucleic acid molecules in accordance with the present inventionmay also be conjugated with radioisotopes, or chemiluminescent,fluorescent, or other labeling compounds (e.g., digoxigenin). Inaddition, the nucleic acid molecules of the present invention may bemodified by nucleic acid modifying enzymes, for example, kinases orphosphatases. These and other modifications of nucleic acid moleculesare well known in the art.

In addition, a nucleic acid molecule that encodes a B7-related factor,or a biologically active fragment thereof, can be ligated to aheterologous sequence to encode a fusion protein (also called a chimericprotein). For example, it may be useful to construct a nucleic acidencoding a fusion protein comprising a B7-related factor and the Fcdomain of human IgG as described herein. The resulting BSL1-Ig, BSL2-Ig,and BSL3-Ig fusion proteins can then be expressed in host cells, andused to prepare pharmaceutical compositions useful for immunomodulation(see below). Fusion proteins comprising B7-related polypeptides can alsobe used for the isolation and purification of B7-related polypeptides orantibodies (see below). In addition, fusion proteins can be used toidentify cellular ligands or binding partners for BSL1, BSL2, or BSL3(see below).

B7-Related Nucleic Acid Expression Vectors

Another aspect of the present invention pertains to expression vectorscomprising a nucleic acid encoding at least one B7-related factor, asdescribed herein, operably linked to at least one regulatory sequence.“Operably linked” is intended to mean that the nucleotide acid sequenceis linked to a regulatory sequence in a manner that allows expression ofthe nucleotide sequence. Regulatory sequences are known in the art andare selected to direct expression of the desired protein in anappropriate host cell. Accordingly, the term regulatory sequenceincludes promoters, enhancers and other expression control elements (seeD. V. Goeddel (1990) Methods Enzymol. 185:3-7). It should be understoodthat the design of the expression vector may depend on such factors asthe choice of the host cell to be transfected and/or the type ofpolypeptide desired to be expressed.

Appropriate host cells for use with the present invention includebacteria, fungi, yeast, plant, insect, and animal cells, especiallymammalian and human cells. Preferred replication and inheritance systemsinclude M13, ColE1, SV40, baculovirus, lambda, adenovirus, CEN ARS, 2 μmARS and the like. Several regulatory elements (e.g., promoters) havebeen isolated and shown to be effective in the transcription andtranslation of heterologous proteins in the various hosts. Suchregulatory regions, methods of isolation, manner of manipulation, etc.are known in the art. Non-limiting examples of bacterial promotersinclude the β-lactamase (penicillinase) promoter; lactose promoter;tryptophan (trp) promoter; araBAD (arabinose) operon promoter;lambda-derived P₁ promoter and N gene ribosome binding site; and thehybrid tac promoter derived from sequences of the trp and lac UV5promoters. Non-limiting examples of yeast promoters include the3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter,galactoepimerase promoter, and alcohol dehydrogenase (ADH1) promoter.Suitable promoters for mammalian cells include, without limitation,viral promoters, such as those from Simian Virus 40 (SV40), Rous sarcomavirus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV).

Eukaryotic cells may also require terminator sequences, polyadenylationsequences, and enhancer sequences that modulate gene expression.Sequences that cause amplification of the gene may also be desirable.These sequences are well known in the art. Furthermore, sequences thatfacilitate secretion of the recombinant product from cells, including,but not limited to, bacteria, yeast, and animal cells, such as secretorysignal sequences and/or preprotein or proprotein sequences, may also beincluded. Such sequences are well described in the art.

Suitable expression vectors include, but are not limited to, pUC,pBluescript (Stratagene), pET (Novagen, Inc., Madison, Wis.), and pREP(Invitrogen) plasmids. Vectors can contain one or more replication andinheritance systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes. The inserted coding sequences can be synthesizedby standard methods, isolated from natural sources, or prepared ashybrids. Ligation of the coding sequences to transcriptional regulatoryelements (e.g., promoters, enhancers, and/or insulators) and/or to otheramino acid encoding sequences can be carried out using establishedmethods.

In one embodiment, the expression vector comprises a nucleic acidencoding at least a portion of the BSL1, BSL2, or BSL3 polypeptide. Inanother embodiment, the expression vector comprises a DNA sequenceencoding the B7-related factor and a DNA sequence encoding anotherB7-related factor or a heterologous polypeptide or peptide. Suchexpression vectors can be used to transfect host cells to therebyproduce polypeptides or peptides, including fusion proteins or peptidesencoded by nucleic acid molecules as described below.

Isolation of B7-Related Polypeptides

Yet another aspect of the present invention pertains to methods ofisolating B7-related polypeptides and related peptides. As used herein,the terms “protein” and “polypeptide” are synonymous. Peptides aredefined as fragments or portions of proteins or polypeptides, preferablyfragments or portions having the same or equivalent function or activityas the complete protein. Both naturally occurring and recombinant formsof the B7-related polypeptides or peptides may be used in assays andtreatments according to the present invention. Methods for directlyisolating and purifying polypeptides or peptides from natural sourcessuch as cellular or extracellular lysates are well known in the art (seeE. L. V. Harris and S. Angal, Eds. (1989) Protein Purification Methods:A Practical Approach, IRL Press, Oxford, England). Such methods include,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, high-performance liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography, andcountercurrent distribution, and combinations thereof. Naturallyoccurring polypeptides can be purified from many possible sources, forexample, plasma, body cells and tissues, or body fluids.

To produce recombinant B7-related polypeptides or peptides, DNAsequences encoding the B7-related polypeptides or peptides are clonedinto a suitable vector for expression in intact host cells or incell-free translation systems (see J. Sambrook et al., supra).Prokaryotic and eukaryotic vectors and host cells may be employed. Theparticular choice of a vector, host cell, or translation system is notcritical to the practice of the invention. DNA sequences can beoptimized, if desired, for more efficient expression in a given hostorganism. For example, codons can be altered to conform to the preferredcodon usage in a given host cell or cell-free translation system usingtechniques routinely practiced in the art.

For some purposes, it may be preferable to produce peptides orpolypeptides in a recombinant system wherein the peptides orpolypeptides carry additional sequence tags to facilitate purification.Such markers include epitope tags and protein tags. Non-limitingexamples of epitope tags include c-myc, haemagglutinin (HA),polyhistidine (6×-HIS) (SEQ ID NO:93), GLU-GLU, and DYKDDDDK (SEQ IDNO:94) (FLAG®) epitope tags. Epitope tags can be added to peptides by anumber of established methods. DNA sequences of epitope tags can beinserted into peptide coding sequences as oligonucleotides or throughprimers used in PCR amplification. As an alternative, peptide-codingsequences can be cloned into specific vectors that create fusions withepitope tags; for example, pRSET vectors (Invitrogen Corp., San Diego,Calif.). Non-limiting examples of protein tags includeglutathione-S-transferase (GST), green fluorescent protein (GFP), andmaltose binding protein (MBP). Protein tags are attached to peptides orpolypeptides by several well-known methods. In one approach, the codingsequence of a polypeptide or peptide can be cloned into a vector thatcreates a fusion between the polypeptide or peptide and a protein tag ofinterest. Suitable vectors include, without limitation, the exemplaryplasmids, pGEX (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.),pEGFP(CLONTECH Laboratories, Inc, Palo Alto, Calif.), and pMAL™ (NewEngland BioLabs, Inc., Beverly, Mass.). Following expression, theepitope or protein tagged polypeptide or peptide can be purified from acrude lysate of the translation system or host cell by chromatography onan appropriate solid-phase matrix. In some cases, it may be preferableto remove the epitope or protein tag (i.e., via protease cleavage)following purification.

Suitable cell-free expression systems for use in accordance with thepresent invention include rabbit reticulocyte lysate, wheat germextract, canine pancreatic microsomal membranes, E. coli S30 extract,and coupled transcription/translation systems (Promega Corp., Madison,Wis.). These systems allow the expression of recombinant polypeptides orpeptides upon the addition of cloning vectors, DNA fragments, or RNAsequences containing coding regions and appropriate promoter elements.

Host cells for recombinant cloning vectors include bacterial,archebacterial, fungal, plant, insect and animal cells, especiallymammalian cells. Of particular interest are E. coli, B. subtilis, S.aureus, S. cerevisiae, S. pombe, N. crassa, SF9, C129, 293, NIH 3T3,CHO, COS, and HeLa cells. Such cells can be transformed, transfected, ortransduced, as appropriate, by any suitable method includingelectroporation, CaCl₂-, LiCl—, LiAc/PEG-, spheroplasting-,Ca-Phosphate, DEAE-dextran, liposome-mediated DNA uptake, injection,microinjection, microprojectile bombardment, or other establishedmethods.

In order to identify host cells that contain the expression vector, agene that contains a selectable marker is generally introduced into thehost cells along with the gene of interest. Preferred selectable markersinclude those that confer resistance to drugs, such as G418, hygromycin,methotrexate, or ampicillin. Selectable markers can be introduced on thesame plasmid as the gene of interest. Host cells containing the gene ofinterest are identified by drug selection, as cells that carry thedrug-resistance marker survive in growth media containing thecorresponding drug.

The surviving cells can be screened for production of recombinantB7-related polypeptides, or peptides or fusions thereof. In oneembodiment, the recombinant polypeptides are secreted to the cellsurface, and can be identified by cell surface staining with ligands tothe B cell antigens (e.g., CD28-Ig). In another embodiment, therecombinant polypeptides are retained in the cytoplasm of the hostcells, and can be identified in cell extracts using anti-B7-relatedpolypeptide antibodies. In yet another embodiment, soluble recombinantpolypeptides are secreted into the growth media, and can be identifiedby screening the growth media with anti-B7-related polypeptideantibodies. A soluble, secreted recombinant B7-polypeptide includes theextracellular domain of the polypeptide, or any fragment thereof, thatdoes not include the cytoplasmic and/or transmembrane regions. Thecell-surface and cytoplasmic recombinant B7-related polypeptides can beisolated following cell lysis and extraction of cellular proteins, whilethe secreted recombinant B7-related polypeptides can be isolated fromthe cell growth media by standard techniques (see I. M. Rosenberg, Ed.(1996) Protein Analysis and Purification: Benchtop Techniques,Birkhauser, Boston, Cambridge, Mass.).

Antibody-based methods can used to purify natural or recombinantlyproduced B7-related polypeptides or peptides. Antibodies that recognizethese polypeptides, or peptides derived therefrom, can be produced andisolated using methods known and practiced in the art (see below).B7-related polypeptides or peptides can then be purified from a crudelysate by chromatography on antibody-conjugated solid-phase matrices(see E. Harlow and D. Lane, 1999, Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Otherpurification methods known and used in the art may also be employed.

It is noted that transfected host cells that express B7-related factors(e.g., BSL1, BSL2, and/or BSL3) or portions thereof on the surface ofthe cell are within the scope of this invention. For example, a tumorcell such as a sarcoma, melanoma, leukemia, lymphoma, carcinoma, orneuroblastoma can be transfected with an expression vector directing theexpression of at least one B7-related factor on the surface of the tumorcell. Such transfected tumor cells can be used to treat tumor immunityas described in detail herein.

B7-Related Polypeptides

A further aspect of the present invention pertains to isolatedB7-related polypeptides. The present invention encompasses the BSL1 (SEQID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15)polypeptides, and fragments and functional equivalents thereof. Suchpolypeptides can comprise at least 5, 12, 20, 30, 50, 100, 170, 200,210, 300, or 500 contiguous amino acid residues. Preferred arepolypeptides that share moderate homology with BSL1 (SEQ ID NO:2), BSL2(SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15) polypeptides. Morepreferred are polypeptides that share substantial homology with BSL1(SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15).

The term “functional equivalent” is intended to include proteins whichdiffer in amino acid sequence from a given B7-related polypeptide, suchas sequence of BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), orBSL3 (SEQ ID NO:15) polypeptide, but where such differences result in amodified protein which performs at least one characteristic function ofthe B7-related polypeptide (e.g., ligand-binding, antigenic, intra- orintercellular activity). For example, a functional equivalent of a BSL1,BSL2, or BSL3 polypeptide may have a modification such as asubstitution, addition or deletion of an amino acid residue which is notdirectly involved in the function of this polypeptide (i.e., the abilityof these polypeptides to co-stimulate T-cell proliferation). Inaddition, non-naturally occurring analogs of B7-related polypeptidescapable of binding CD28 and/or CD28-related ligand(s) are consideredfunctional equivalents. Various modifications of the B7-relatedpolypeptides to produce functional equivalents of these polypeptides aredescribed in detail herein.

It is also possible to modify the structure of a B7-related polypeptidefor such purposes as increasing solubility, enhancing therapeutic orprophylactic efficacy (reactivity), or stability (e.g., shelf life exvivo and resistance to proteolytic degradation in vivo). Such modifiedproteins are considered functional equivalents of the B7-relatedpolypeptides as defined herein. Preferably, the B7-related polypeptidesare modified so that they retain the ability to co-stimulate T-cellproliferation. Those residues shown to be essential to interact with theCD28 or CD28-related ligands on T-cells can be modified by replacing theessential amino acid with another, preferably similar amino acid residue(a conservative substitution) whose presence is shown to enhance,diminish, but not eliminate, or not effect receptor interaction. Inaddition, those amino acid residues that are not essential for receptorinteraction can be modified by being replaced by another amino acidwhose incorporation may enhance, diminish, or not effect reactivity. Forexample, a B7-related polypeptide can be modified by substitution ofcysteine residues with other amino acids, such as alanine, serine,threonine, leucine, or glutamic acid, to prevent dimerization viadisulfide linkages. In addition, the amino acid side chains of aB7-related polypeptide of the invention can be chemically modified.Also, a B7-related polypeptide can be modified by cyclization of theamino acid sequence.

In order to enhance stability and/or reactivity, the B7-relatedpolypeptides can be altered to incorporate one or more polymorphisms inthe amino acid sequence. Additionally, D-amino acids, non-natural aminoacids, or non-amino acid analogs can be substituted or added to producea modified polypeptide. Furthermore, the B7-related polypeptidesdisclosed herein can be modified using polyethylene glycol (PEG)according to known methods (Wie et al., supra) to produce a proteinconjugated with PEG. In addition, PEG can be added during chemicalsynthesis of the protein. Other possible modifications includereduction/alkylation (Tarr (1986) Methods of ProteinMicrocharacterization, J. E. Silver, Ed., Humana Press, Clifton, N.J.,pp. 155-194); acylation (Tarr, supra); chemical coupling to anappropriate carrier (Mishell and Shiigi, Eds. (1980) Selected Methods inCellular Immunology, W H Freeman, San Francisco, Calif.; U.S. Pat. No.4,939,239; or mild formalin treatment (Marsh (1971) Int. Arch. ofAllergy and Appl. Immunol. 41:199-215) of the B7-related polypeptide.

Modified polypeptides can have conservative changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. More infrequently, amodified polypeptide can have non-conservative changes, e.g.,substitution of a glycine with a tryptophan. Guidance in determiningwhich amino acid residues can be substituted, inserted, or deletedwithout abolishing biological or immunological activity can be foundusing computer programs well known in the art, for example, DNASTARsoftware (DNASTAR, Inc., Madison, Wis.)

As non-limiting examples, conservative substitutions in the B7-relatedamino acid sequence can be made in accordance with the following table:

Original Conservative Residue Substitution(s) Ala Ser Arg Lys Asn Gln,His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, ValLeu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser ThrThr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunogenicity can be made byselecting substitutions that are less conservative than those shown inthe table, above. For example, non-conservative substitutions can bemade which more significantly affect the structure of the polypeptide inthe area of the alteration, for example, the alpha-helical, orbeta-sheet structure; the charge or hydrophobicity of the molecule atthe target site; or the bulk of the side chain. The substitutions whichgenerally are expected to produce the greatest changes in thepolypeptide's properties are those where 1) a hydrophilic residue, e.g.,seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; 2) a cysteineor proline is substituted for (or by) any other residue; 3) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or 4) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) a residue that does not have aside chain, e.g., glycine.

Preferred polypeptide embodiments further include an isolatedpolypeptide comprising an amino acid sequence sharing at least 60, 70,80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with an amino acidsequence of BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3(SEQ ID NO:15). This polypeptide sequence may be identical to thesequence of BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3(SEQ ID NO:15), or may include up to a certain integer number of aminoacid alterations as compared to the reference sequence

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTPand TBLASTN programs are publicly available from NCBI and other sources.The well-known Smith Waterman algorithm may also be used to determineidentity.

Exemplary parameters for amino acid sequence comparison include thefollowing: 1) algorithm from Needleman and Wunsch, 1970, J. Mol. Biol.48:443-453; 2) BLOSSUM62 comparison matrix from Hentikoff and Hentikoff,1992, Proc. Natl. Acad. Sci. USA 89:10915-10919; 3) gap penalty=12; and4) gap length penalty=4. A program useful with these parameters ispublicly available as the “gap” program (Genetics Computer Group,Madison, Wis.). The aforementioned parameters are the default parametersfor polypeptide comparisons (with no penalty for end gaps).

Alternatively, polypeptide sequence identity can be calculated using thefollowing equation: % identity=(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps.

In accordance with the present invention, polypeptide sequences may beidentical to the sequence of BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQID NO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15), or may include up toa certain integer number of amino acid alterations. Polypeptidealterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion. Alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. In specificembodiments, polypeptide variants may be encoded by BSL1, BSL2, or BSL3nucleic acids comprising single nucleotide polymorphisms and/oralternate splice variants. Polypeptides may also be modified by, forexample, phosphorylation, sulfation, or acylation. They may also bemodified with a label capable of providing a detectable signal, eitherdirectly or indirectly, including, but not limited to, radioisotopes andfluorescent compounds.

The invention also relates to isolated, synthesized and/or recombinantportions or fragments of a BSL1 (e.g., SEQ ID NO:2), BSL2 (e.g., SEQ IDNO:7, 11, or 13), or BSL3 (e.g., SEQ ID NO:15) protein or polypeptide asdescribed herein. Polypeptide fragments (i.e., peptides) can be madewhich have full or partial function on their own, or which when mixedtogether (though fully, partially, or nonfunctional alone),spontaneously assemble with one or more other polypeptides toreconstitute a functional protein having at least one functionalcharacteristic of a BSL1, BSL2, or BSL3 protein of this invention. Inaddition, B7-related polypeptide fragments may comprise, for example,one or more domains of the polypeptide (e.g., the transmembrane orextracellular domain) disclosed herein.

The polypeptides of the present invention, includingfunction-conservative variants, may be isolated from wild-type or mutantcells (e.g., human cells or cell lines), from heterologous organisms orcells (e.g., bacteria, yeast, insect, plant, and mammalian cells), orfrom cell-free translation systems (e.g., wheat germ, microsomalmembrane, or bacterial extracts) in which a protein-coding sequence hasbeen introduced and expressed. Furthermore, the polypeptides may be partof recombinant fusion proteins. The polypeptides can also,advantageously, be made by synthetic chemistry. Polypeptides may bechemically synthesized by commercially available automated procedures,including, without limitation, exclusive solid phase synthesis, partialsolid phase methods, fragment condensation or classical solutionsynthesis. Both the naturally occurring and recombinant forms of thepolypeptides of the invention can advantageously be used to screencompounds for binding activity. The polypeptides of the invention alsofind use as therapeutic agents as well as antigenic components toprepare antibodies as described in detail herein.

Antibodies to B7-Related Polypeptides

Another aspect of the present invention encompasses antibodies thatspecifically recognize B7-related polypeptides or peptides, preferablythe BSL1, BSL2, or BSL3 polypeptides, or fragments derived therefrom. Asused herein, “antibody” refers to intact molecules as well as fragmentsthereof, such as Fab, F(ab)₂, and Fv, which are capable of binding anepitopic determinant. Antibodies that bind to a B7-related polypeptide,preferably the BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), orBSL3 (SEQ ID NO:15) polypeptide, can be prepared using the isolatedB7-related polypeptide or peptide fragments as the immunogen orimmunizing antigen by methods known in the art (see I. Lefkovits, Ed.,(1996) Immunology Methods Manual Academic Press, Inc., San Diego,Calif.). As will be appreciated by those having skill in the art, theimmunogen can be conjugated to a carrier protein, if desired, toincrease immunogenicity, particularly, if a small peptide is used.Commonly used carriers that are routinely used chemically coupled topeptides include serum albumins, e.g., bovine, sheep, goat, or fishserum albumin, thyroglobulin, and keyhole limpet hemocyanin. The coupledimmunogen-carrier is then used to immunize a recipient animal (e.g.,mouse, rat, sheep, goat, or rabbit).

The term “antigenic determinant” refers to that fragment of a molecule(i.e., an epitope) that makes contact with a particular antibody. Whenthe B7-polypeptide or peptide fragment is used to immunize a hostanimal, numerous regions of the polypeptide or peptide fragment mayinduce the production of antibodies which bind specifically to a givenregion or three-dimensional structure on the polypeptide or peptide.Such regions or structures are referred to as antigenic determinants orepitopes. An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody. Preferred are those antigenic determinants that arespecific for the BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), orBSL3 (SEQ ID NO:15) polypeptide or peptide. B7-related polypeptide- orpeptide-derived immunogens can be obtained using techniques as describedabove. For example, B7-related polypeptide or peptide fragments can beisolated from natural or recombinant sources and purified by excisingthe polypeptides or peptide fragments from gels, particularly SDS-PAGEgels.

BSL1-, BSL2-, or BSL3-specific antibodies according to the presentinvention include polyclonal and monoclonal antibodies. The antibodiescan be elicited in an animal host by immunization with B7-relatedpolypeptide-derived immunogenic components or can be formed by in vitroimmunization (sensitization) of immune cells. The immunogenic componentsused as immunogens to elicit the production of antibodies can beisolated from plasma, recombinantly produced, or chemically synthesized.The antibodies can also be produced in recombinant systems programmedwith appropriate antibody-encoding DNA. Alternatively, the antibodiescan be constructed by biochemical reconstitution of purified heavy andlight chains. The antibodies include hybrid antibodies, chimericantibodies, humanized antibodies (see, for example, U.S. Pat. No.5,585,089 to C. J. Queen et al.) and univalent antibodies. Also includedare Fab fragments, Fab′ and F(ab)₂ fragments of antibodies. Notably,antibodies that specifically recognize either the secreted, cell surfaceform, or the cytoplasmic, soluble form of a B7-related factor can beisolated.

Hybridomas that produce monoclonal antibodies against the immunogeniccomponents of the invention can be produced by well-known techniques.Hybridomas can be produced by the fusion of an immortalized cell linewith a B-lymphocyte that produces the desired antibody. Alternatively,non-fusion techniques for generating immortal antibody-producing celllines are possible, and are within the purview of the present invention(see Casali et al. (1986) Science 234:476). Immortalized cell lines aretypically transformed mammalian cells, particularly myeloma cells ofrodent, bovine, and human origin. Most frequently, rat or mouse myelomacell lines are employed as a matter of convenience and availability.Standard procedures can be used to select hybridomas, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Hybridomas that secretedesired monoclonal antibodies can be selected by assaying the cells'culture medium by standard immunoassays, such as immunoblotting, ELISA(enzyme-linked immunosorbent assay; E. Engvall et al. (1971)Immunochemistry, 8:871-4; and D. J. Reen (1994) Methods Mol. Biol.32:461-6), RIA (radioimmunoassay), or comparable assays. Antibodies canbe recovered from the medium using standard protein purificationtechniques (see Tijssen (1985) Practice and Theory of EnzymeImmunoassays, Elsevier, Amsterdam).

In one embodiment, antibodies that react with a B7-related polypeptideor peptide fragment are used in accordance with the present invention toidentify or isolate a B7-related polypeptide or peptide fragment in abiological sample. To isolate a B7-related polypeptide from a sample,antibodies that specifically recognize and bind to a B7-relatedpolypeptide or peptide fragment are conjugated to a solid support, theantibody-conjugated solid support is incubated with the sample or analiquot of the sample, and the polypeptide or peptide that binds to theantibodies is eluted from the solid support. To detect a B7-relatedpolypeptide or peptide fragment in a sample, the sample is incubatedwith antibodies that specifically recognize and bind to a B7-relatedpolypeptide or peptide fragment under conditions that allow theantibodies to bind to the polypeptide or peptide fragment, and thebinding of the antibodies to the B7-related polypeptide or peptidefragment is determined.

Assays Utilizing B7-Related Nucleic Acids or Polypeptides

Expression Analysis of B7-Related Factors:

Several well-established techniques can be used to determine theexpression levels, patterns, and cell-type specificity of the B7-relatedfactors. For example, mRNA levels can be determined utilizing northernblot analysis (J. C. Alwine et al. (1977) Proc. Natl. Acad. Sci. USA74:5350-5354; I. M. Bird (1998) Methods Mol. Biol. 105:325-36), wherebypoly(A)⁺ RNA is isolated from cells, separated by gel electrophoresis,blotted onto a support surface (e.g., nitrocellulose or Immobilon-Ny+(Millipore Corp., Bedford, Mass.)), and incubated with a labeled (e.g.,fluorescently labeled or radiolabeled) oligonucleotide probe that iscapable of hybridizing with the mRNA of interest. Alternatively, mRNAlevels can be determined by quantitative (for review, see W. M. Freemanet al. (1999) Biotechniques 26:112-122) or semi-quantitative RT-PCRanalysis (Ren et al. Mol. Brain. Res. 59:256-63). In accordance withthis technique, poly(A)⁺ RNA is isolated from cells, used for cDNAsynthesis, and the resultant cDNA is incubated with PCR primers that arecapable of hybridizing with the template and amplifying the templatesequence to produce levels of the PCR product that are proportional tothe cellular levels of the mRNA of interest. Another technique, in situhybridization, can also be used to determine mRNA levels (reviewed by A.K. Raap (1998) Mutat. Res. 400:287-298). In situ hybridizationtechniques allow the visual detection of mRNA in a cell by incubatingthe cell with a labeled (e.g., fluorescently labeled or digoxigeninlabeled) oligonucleotide probe that hybridizes to the mRNA of interest,and then examining the cell by microscopy.

Chromosomal Mapping of B7-Related Genes:

The chromosomal location of B7-related genes can be determined byvarious techniques known in the art. For example, high-resolutionchromosomal banding can be used (reviewed by M. Ronne (1990) In Vivo4:337-65). High-resolution banding techniques utilize elongatedchromosomes from cells at early mitotic stages, which have beensynchronized using DNA-synthesis inhibitors (e.g., methotrexate orthymidine) or DNA-binding agents (e.g., ethidium bromide). However,these techniques can only be used to map a gene to a relatively largeregion of a chromosome (˜3 Mb). For more accurate gene mapping,fluorescence in situ hybridization (FISH) techniques can be used. Inparticular, high-resolution FISH techniques (A. Palotie et al. (1996)Ann. Med. 28:101-106) utilize free chromatin, DNA fibers, ormechanically-stretched chromosomes to map gene sequences ranging fromseveral kilobases to 300 kb in size. Alternatively, the chromosomallocation of a gene can be determined from the appropriate genomedatabase, for example, the Homo sapiens genome database available at theEntrez Genome website (National Center for Biotechnology Information,Bethesda, Md.)

Identification of T-Cell Ligands:

The B7-related polypeptides or peptides disclosed herein can be used toidentify their cognate ligands on immune or inflammatory response cells,such as T-cells (i.e., CD28- or CTLA-4-related ligands). Candidateligands, or fragments derived therefrom, can be identified and analyzedby many well-known methods in the art (see T. E. Creighton, Ed., 1997,Proteins Structure: A Practical Approach, IRL Press at Oxford Press,Oxford, England). For example, T-cell ligands that bind to theB7-related polypeptides or peptides can be identified from extracts orlysates obtained from animal, preferably human, immune or inflammatoryresponse cells (e.g., T-cells). The proteins obtained from these sourcescan be separated into bands using sodium dodecylsulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and transferred by electroblotting, forexample, onto a suitable solid-phase support or membrane (e.g.,nitrocellulose or polyvinylidene fluoride (PVDF)). The solid-phasesupport or membrane can then be incubated with a labeled form of aB7-related polypeptide or peptide, e.g., BSL1, BSL2, or BSL3 proteinsthat correspond to bands that exhibit specific binding with the labeledB7-related polypeptide or peptide can then be identified, isolated,purified, and analyzed by amino acid analysis and/or Edman degradationto determine the amino acid sequence of peptides derived therefrom.

As an alternative approach, a fusion protein comprising a B7-relatedpolypeptide can be attached to a solid support and incubated withextracts obtained from cells, such as CHO or COS cells, that aretransfected with an appropriate cDNA library. For example, a cDNAlibrary can be constructed from resting or activated immortal humanT-cell lines, such as CEM, HUT78, or Jurkat cell lines, or from restingor activated human T-cells derived from peripheral blood, tonsil,spleen, thymus or other specialized lymphoid tissues. Such cells can beactivated by the addition of anti-CD3 and anti-CD28 monoclonalantibodies, phytohemaglutinin (PHA), or phorbol 12-myristate-13-acetate(PMA) with ionomycin. The cDNA library construct can contain a removableepitope tag (see above) that is different from the fusion protein, andwill facilitate purification of the library expression product(s) thatassociate with the fusion protein. The isolated library expressionproduct(s) can then be isolated and characterized. In addition, a fusionprotein comprising a B7-related polypeptide can be attached to a solidsupport (e.g., a column comprising beads that specifically bind to thefusion protein) and incubated with lysates obtained from cells, such asT-cells, that are enriched for integral membrane proteins. The cellularproteins that associate with the fusion protein can be isolated and thencharacterized using MALDI-TOF analysis (Matrix Assisted Laser DesorptionIonization Time Of Flight Analysis; reviewed by Yates J R 3rd. (1998) J.Mass Spectrom. 33:1-19; P. Chaurand et al. (1999) J. Am. Soc. MassSpectrom. 10:91-103). Fusion proteins can include, for example,FLAG®—(B. L. Brizzard et al. (1994) Biotechniques 16:730-735), 6×-HIS,and GST-tagged fusion proteins (see above), which can be attached tosolid supports that are conjugated with anti-FLAG® antibodies, nickel,or glutathione molecules, respectively. Methods of producing andpurifying such fusion proteins are well known in the art.

Another suitable ligand-binding assay is the yeast two-hybrid system(Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,283,173). Thetwo-hybrid system relies on the reconstitution of transcriptionactivation activity by association of the DNA-binding and transcriptionactivation domains of a transcriptional activator throughprotein-protein interaction. The yeast GAL4 transcriptional activatormay be used in this way, although other transcription factors have beenused and are well known in the art. To carryout the two-hybrid assay,the GAL4 DNA-binding domain and the GAL4 transcription activation domainare expressed, separately, as fusions to potential interactingpolypeptides. For example, one fusion protein can comprise a B7-relatedpolypeptide fused to the GAL4 DNA-binding domain. The other fusionprotein can comprise, for example, a T-cell cDNA library encodedpolypeptide fused to the GAL4 transcription activation domain. If thetwo, coexpressed fusion proteins interact in the nucleus of a host cell,a reporter gene (e.g. LacZ) is activated to produce a detectablephenotype. The host cells that show two-hybrid interactions can be usedto isolate the containing plasmids containing the cDNA librarysequences. These plasmids can be analyzed to determine the nucleic acidsequence and predicted polypeptide sequence of the candidate T-cellligand.

Related, in vivo, methods such as the three-hybrid (Licitra et al.(1996) Proc. Natl. Acad. Sci. USA 93:12817-12821), and reversetwo-hybrid (Vidal et al. (1996) Proc. Natl. Acad. Sci. USA93:10315-10320) systems may serve as alternative approaches.Commercially available two-hybrid systems such as the CLONTECHMATCHMAKER™ systems and protocols (CLONTECH, Palo Alto, Calif.) may bealso be used. (See also, A. R. Mendelsohn et al. (1994) Curr. Op.Biotech. 5:482; E. M. Phizicky et al. (1995) Microbiological Rev. 59:94;M. Yang et al. (1995) Nucleic Acids Res. 23:1152; S. Fields et al.(1994) Trends Genet. 10:286; and U.S. Pat. Nos. 6,283,173 and5,468,614).

Ligand sequence(s) obtained from ligand-binding assay(s) can be comparedwith subject sequences in available databases such as, withoutlimitation, GenPept, SWISS-PROT®, and Incyte Genomics databases (IncyteGenomics). These databases, which contain previously identified andannotated sequences, may be searched for the full-length polypeptide andgene sequence using, for example, BLAST analysis (see above). In caseswhere the full-length sequences of the ligands are not available,extended or overlapping partial clones may be obtained by techniquesconventionally known and practiced in the art. Non-limiting examples ofsuch techniques include hybridization to plasmid or phage libraries ofgenomic DNA or cDNA; PCR from the same libraries using B7-related factorprimer pairs; or hybridization or PCR directly to genomic DNA or cDNA.These clones may then be sequenced and assembled into full-length genesusing the fragment sequence alignment program (PHRAP; Nickerson et al.(1997) Nucleic Acids Res. 25:2745-2751).

Assays for B7-Related Factor Activity:

Screening the fragments, mutants or variants for those which retaincharacteristic B7-related polypeptide activity as described herein canbe accomplished using one or more of several different assays. Forexample, appropriate cells, such as CHO cells, can be transfected withthe cloned variants and then analyzed for cell surface phenotype byindirect immunofluorescence and flow cytometry. Cell surface expressionof the transfected cells is evaluated using a monoclonal antibodyspecifically reactive with a cell surface form of a B7-related factor(see above). Production of secreted forms of the B7-related factors canbe evaluated by immunoprecipitation using a monoclonal antibodyspecifically reactive with a B7-related factor.

Other, more preferred, assays take advantage of the functionalcharacteristics of the B7-related factors. As previously set forth, thebinding of the B7-related factors to its T-cell ligand(s) causes thecells to produce increased levels of lymphokines, particularly ofinterleukin-2. Thus, B7-related factor function can be assessed bymeasuring the synthesis of lymphokines, such as interleukin-2 or othernovel and as yet undefined cytokines, and/or assaying for T-cellproliferation by CD28⁺ T-cells that have received a primary activationsignal. Any one of several conventional assays for interleukin-2 can beemployed (see C. B. Thompson (1989) Proc. Natl. Acad. Sci. USA 86:1333).

The same basic functional assays can also be used to screen forB7-related polypeptides, peptides, fusion proteins, or antibodies thatblock T-cell activation. The ability of such proteins to block thenormal costimulatory signal and induce a state of anergy can bedetermined using subsequent attempts at stimulation of the T-cells withantigen presenting cells that express cell surface B cell activationantigen B7 and present antigen. If the T-cells are unresponsive to theactivation attempts, as determined by IL-2 synthesis and T-cellproliferation, a state of anergy has been induced and can be determinedby methods known in the art (see R. H. Schwartz (1990) Science248:1349-1356).

Modulators of B7-Related Factors

The BSL1, BSL2, and BSL3 polypeptides, polynucleotides, variants, orfragments thereof, can be used to screen for test agents (e.g.,agonists, antagonists, or inhibitors) that modulate the levels oractivity of the corresponding B7-related polypeptide. In addition,B7-related molecules can be used to identify endogenous modulators thatbind to BSL1, BSL2, or BSL3 polypeptides or polynucleotides in the cell.In one aspect of the present invention, the full-length BSL1 (e.g., SEQID NO:2), BSL2 (e.g., SEQ ID NO:7, 11, or 13), or BSL3 (e.g., SEQ IDNO:15) polypeptide is used to identify modulators. Alternatively,variants or fragments of a BSL1, BSL2, or BSL3 polypeptide are used.Such fragments may comprise, for example, one or more domains of theB7-related polypeptide (e.g., the extracellular and transmembranedomains) disclosed herein. Of particular interest are screening assaysthat identify agents that have relatively low levels of toxicity inhuman cells. A wide variety of assays may be used for this purpose,including in vitro protein-protein binding assays, electrophoreticmobility shift assays, immunoassays, and the like.

The term “modulator” as used herein describes any test agent, molecule,protein, peptide, or compound with the capability of directly orindirectly altering the physiological function, stability, or levels ofthe BSL1, BSL2, and BSL3 polypeptide. Modulators that bind to theB7-related polypeptides or polynucleotides of the invention arepotentially useful in diagnostic applications and/or pharmaceuticalcompositions, as described in detail herein. Test agents useful asmodulators may encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons. Suchmolecules can comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. Test agentswhich can be used as modulators often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Test agentscan also comprise biomolecules including peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogs,or combinations thereof.

Test agents finding use as modulators may include, for example, 1)peptides such as soluble peptides, including Ig-tailed fusion peptidesand members of random peptide libraries (see, e.g., Lam et al. (1991)Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang et al., (1993) Cell 72:767-778); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules.

Test agents and modulators can be obtained from a wide variety ofsources including libraries of synthetic or natural compounds. Syntheticcompound libraries are commercially available from, for example,Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton,N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (NewMilford, Conn.). A rare chemical library is available from AldrichChemical Company, Inc. (Milwaukee, Wis.). Natural compound librariescomprising bacterial, fungal, plant or animal extracts are availablefrom, for example, Pan Laboratories (Bothell, Wash.). In addition,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts can be readily produced. Methods forthe synthesis of molecular libraries are readily available (see, e.g.,DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al.(1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J.Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.37:1233). In addition, natural or synthetic compound libraries andcompounds can be readily modified through conventional chemical,physical and biochemical means (see, e.g., Blondelle et al. (1996)Trends in Biotech. 14:60), and may be used to produce combinatoriallibraries. In another approach, previously identified pharmacologicalagents can be subjected to directed or random chemical modifications,such as acylation, alkylation, esterification, amidification, and theanalogs can be screened for BSL1-, BSL2-, and BSL3-modulating activity.

Numerous methods for producing combinatorial libraries are known in theart, including those involving biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topolypeptide libraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer, or small molecule libraries ofcompounds (K. S. Lam, 1997, Anticancer Drug Des. 12:145).

Libraries may be screened in solution (e.g., Houghten, (1992)Biotechniques 13:412-421), or on beads (Lam, (1991) Nature 354:82-84),chips (Fodor, (1993) Nature 364:555-556), bacteria or spores (LadnerU.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad.Sci. USA 89:1865-1869), or on phage (Scott and Smith, (1990) Science249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al. (1990)Proc. Natl. Acad. Sci. USA 97:6378-6382; Felici, (1991) J. Mol. Biol.222:301-310; Ladner, supra).

Where the screening assay is a binding assay, a BSL1, BSL2, and BSL3polypeptide, polynucleotide, analog, or fragment thereof, may be joinedto a label, where the label can directly or indirectly provide adetectable signal. Various labels include radioisotopes, fluorescers,chemiluminescers, enzymes, specific binding molecules, particles, e.g.magnetic particles, and the like. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin, etc.For the specific binding members, the complementary member wouldnormally be labeled with a molecule that provides for detection, inaccordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc., that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc., may be used. Thecomponents are added in any order that produces the requisite binding.Incubations are performed at any temperature that facilitates optimalactivity, typically between 4° and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Normally, between 0.1 and 1 hr (hour)will be sufficient. In general, a plurality of assay mixtures is run inparallel with different agent concentrations to obtain a differentialresponse to these concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection.

To perform cell-free screening assays, it may be desirable to immobilizeeither the BSL1, BSL2, or BSL3 polypeptide, polynucleotide, or fragmentto a surface to facilitate identification of modulators that bind tothese molecules, as well as to accommodate automation of the assay. Forexample, a fusion protein comprising a BSL1, BSL2, or BSL3 polypeptideand an affinity-tag can be produced as described in detail herein. Inone embodiment, a GST-fusion protein comprising a BSL1, BSL2, or BSL3polypeptide is adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates.Cell lysates (e.g., containing ³⁵S-labeled polypeptides) are added tothe polypeptide-coated beads under conditions to allow complex formation(e.g., at physiological conditions for salt and pH). Followingincubation, the polypeptide-coated beads are washed to remove anyunbound polypeptides, and the amount of immobilized radiolabel isdetermined. Alternatively, the complex is dissociated and the radiolabelpresent in the supernatant is determined. In another approach, the beadsare analyzed by SDS-PAGE to identify BSL1-, BSL2-, or BSL3-bindingpolypeptides.

Various binding assays can be used to identify agonist or antagoniststhat alter the function or levels of the BSL1, BSL2, or BSL3polypeptide. Such assays are designed to detect the interaction of testagents with BSL1, BSL2, or BSL3 polypeptides, polynucleotides,functional equivalents, or fragments thereof. Interactions may bedetected by direct measurement of binding. Alternatively, interactionsmay be detected by indirect indicators of binding, such asstabilization/destabilization of protein structure, oractivation/inhibition of biological function. Non-limiting examples ofuseful binding assays are detailed below.

Modulators that bind to BSL1, BSL2, or BSL3 polypeptides,polynucleotides, functional equivalents, or fragments thereof, can beidentified using real-time Bimolecular Interaction Analysis (BIA;Sjolander et al. (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705; e.g., BIAcore™; LKB Pharmacia,Sweden). Modulators can also be identified by scintillation proximityassays (SPA, described in U.S. Pat. No. 4,568,649). Binding assays usingmitochondrial targeting signals (Hurt et al. (1985) EMBO J. 4:2061-2068;Eilers and Schatz, (1986) Nature 322:228-231) a plurality of definedpolymers synthesized on a solid substrate (Fodor et al. (1991) Science251:767-773) may also be employed.

Two-hybrid systems may be used to identify modulators (see, e.g., U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO 94/10300). Alternatively, three-hybrid (Licitra et al.(1996) Proc. Natl. Acad. Sci. USA 93:12817-12821), and reversetwo-hybrid (Vidal et al. (1996) Proc. Natl. Acad. Sci. USA93:10315-10320) systems may be used. Commercially available two-hybridsystems such as the CLONTECH MATCHMAKER™ systems and protocols (CLONTECHLaboratories, Inc., Palo Alto, Calif.) are also useful (see also, A. R.Mendelsohn et al. (1994) Curr. Op. Biotech. 5:482; E. M. Phizicky et al.(1995) Microbiological Rev. 59:94; M. Yang et al. (1995) Nucleic AcidsRes. 23:1152; S. Fields et al. (1994) Trends Genet. 10:286; and U.S.Pat. Nos. 6,283,173 and 5,468,614).

Several methods of automated assays have been developed in recent yearsso as to permit screening of tens of thousands of test agents in a shortperiod of time. High-throughput screening methods are particularlypreferred for use with the present invention. The binding assaysdescribed herein can be adapted for high-throughput screens, oralternative screens may be employed. For example, continuous format highthroughput screens (CF-HTS) using at least one porous matrix allows theresearcher to test large numbers of test agents for a wide range ofbiological or biochemical activity (see U.S. Pat. No. 5,976,813 toBeutel et al.). Moreover, CF-HTS can be used to perform multi-stepassays.

Diagnostics

According to another embodiment of the present invention, the B7-relatedpolynucleotides, or fragments thereof, may be used for diagnosticpurposes. The B7-related polynucleotides that may be used includeoligonucleotide sequences, complementary RNA and DNA molecules, andPNAs. The polynucleotides may be used to detect and quantify levels ofBSL1, BSL2, and BSL3 mRNA in biological samples in which expression (orunder- or overexpression) of BSL1, BSL2, and BSL3 polynucleotide may becorrelated with disease. The diagnostic assay may be used to distinguishbetween the absence, presence, increase, and decrease of the expressionof BSL1, BSL2, and BSL3, and to monitor regulation of BSL1, BSL2, andBSL3 polynucleotide levels during therapeutic treatment or intervention.

In one aspect, PCR probes can be used to detect B7-relatedpolynucleotide sequences, including BSL1, BSL2, and BSL3 genomic DNAsequences and BSL1-, BSL2-, and BSL3-related nucleic acid sequences. Thespecificity of the probe, whether it is made from a highly specificregion, e.g., at least 8 to 10 or 12 or 15 contiguous nucleotides in the5′ regulatory region, or a less specific region, e.g., especially in the3′ coding region, and the stringency of the hybridization oramplification (maximal, high, intermediate, or low) will determinewhether the probe identifies only naturally occurring sequences encodingthe B7-related polypeptide, alleles thereof, or related sequences.

Probes may also be used for the detection of BSL1-, BSL2-, andBSL3-related sequences, and should preferably contain at least 60%,preferably greater than 90%, identity to the BSL1, BSL2, and BSL3polynucleotide, or a complementary sequence, or fragments thereof. Theprobes of this invention may be DNA or RNA, the probes may comprise allor a fragment of the nucleotide sequence of BSL1 (SEQ ID NO:2), BSL2(SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15), or a complementarysequence thereof, and may include promoter, enhancer elements, andintrons of the naturally occurring BSL1, BSL2, or BSL3 polynucleotide.

Methods for producing specific probes for B7-related polynucleotidesinclude the cloning of nucleic acid sequences of BSL1 (SEQ ID NO:2),BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15), or a fragmentthereof, into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of detector/reportergroups, e.g., radionucleotides such as ³²P or ³⁵S, or enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

A wide variety of labels and conjugation techniques are known andemployed by those skilled in the art and may be used in various nucleicacid and amino acid assays. Means for producing labeled hybridization orPCR probes for detecting sequences related to polynucleotides encodingthe BSL1, BSL2, or BSL3 polypeptide include oligo-labeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide. Alternatively, BSL1, BSL2, or BSL3 polynucleotide sequences,or any portions or fragments thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase, such as T7, T3, orSP(6) and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (e.g., from Amersham PharmaciaBiotech, Inc., Piscataway, N.J.; Promega Corp., Madison Wis.; and U.S.Biochemical Corp., U.S. Biochemical Amersham, Cleveland, Ohio). Suitablereporter molecules or labels which may be used include radionucleotides,enzymes, fluorescent, chemiluminescent, or chromogenic agents, as wellas substrates, cofactors, inhibitors, magnetic particles, and the like.

B7-related polynucleotide sequences, or fragments, or complementarysequences thereof, can be used in Southern or Northern analysis, dotblot, or other membrane-based technologies; in PCR technologies; or indip stick, pin, ELISA or biochip assays utilizing fluids or tissues frompatient biopsies to detect the status of, e.g., levels or overexpressionof BSL1, BSL2, or BSL3, or to detect altered BSL1, BSL2, or BSL3expression. Such qualitative or quantitative methods are well known inthe art (G. H. Keller and M. M. Manak, 1993, DNA Probes, 2^(nd) Ed,Macmillan Publishers Ltd., England; D. W. Dieffenbach and G. S.Dveksler, 1995, PCR Primer: A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y.; B. D. Hames and S. J. Higgins, 1985, Gene Probes1, 2, IRL Press at Oxford University Press, Oxford, England).

BSL1, BSL2, and BSL3 oligonucleotides may be chemically synthesized,generated enzymatically, or produced from a recombinant source.Oligomers will preferably comprise two nucleotide sequences, one with asense orientation (5′→3′) and another with an antisense orientation(3′→5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantification of closelyrelated DNA or RNA sequences.

Methods suitable for quantifying the expression of B7-related factorsinclude radiolabeling or biotinylating nucleotides, co-amplification ofa control nucleic acid, and standard curves onto which the experimentalresults are interpolated (P. C. Melby et al. (1993) J. Immunol. Methods159:235-244; and C. Duplaa et al. (1993) Anal. Biochem. 229-236). Thespeed of quantifying multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantification.

In a particular aspect, a nucleic acid sequence complementary to aB7-related polynucleotide, or fragment thereof, may be useful in assaysthat detect diseases relating to aberrant immune responses, particularlythose described herein. A BSL1, BSL2, and/or BSL3 polynucleotide can belabeled by standard methods, and added to a biological sample from asubject under conditions suitable for the formation of hybridizationcomplexes. After a suitable incubation period, the sample can be washedand the signal is quantified and compared with a standard value. If theamount of signal in the test sample is significantly altered from thatof a comparable negative control (normal) sample, the altered levels ofBSL1, BSL2, and/or BSL3 nucleotide sequence can be correlated with thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular prophylactic or therapeuticregimen in animal studies, in clinical trials, or for an individualpatient.

To provide a basis for the diagnosis of a disease associated withaltered expression of one or more B7-related factors, a normal orstandard profile for expression is established. This may be accomplishedby incubating biological samples taken from normal subjects, eitheranimal or human, with a sequence complementary to a BSL1, BSL2, BSL3polynucleotide, or a fragment thereof, under conditions suitable forhybridization or amplification. Standard hybridization may be quantifiedby comparing the values obtained from normal subjects with those from anexperiment where a known amount of a substantially purifiedpolynucleotide is used. Standard values obtained from normal samples maybe compared with values obtained from samples from patients who aresymptomatic for the disease. Deviation between standard and subject(patient) values is used to establish the presence of the condition.

Once the disease is diagnosed and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in a normal individual. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to diseases involving a hyperactive or hypoactive immuneresponse, the presence of an abnormal levels (decreased or increased) ofB7-related transcript in a biological sample (e.g., body fluid, cells,tissues, or cell or tissue extracts) from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier, thereby preventing the development or furtherprogression of the disease.

In one particular aspect, BSL1, BSL2, and BSL3 oligonucleotides may beused for PCR-based diagnostics. For example, PCR can be used to performGenetic Bit Analysis (GBA) of BSL1, BSL2, and/or BSL3 in accordance withpublished methods (T. T. Nikiforov et al. (1994) Nucleic Acids Res.22(20):4167-75; T. T. Nikiforov et al. (1994) PCR Methods Appl.3(5):285-91). In PCR-based GBA, specific fragments of genomic DNAcontaining the polymorphic site(s) are first amplified by PCR using oneunmodified and one phosphorothioate-modified primer. The double-strandedPCR product is rendered single-stranded and then hybridized toimmobilized oligonucleotide primer in wells of a multi-well plate.Notably, the primer is designed to anneal immediately adjacent to thepolymorphic site of interest. The 3′ end of the primer is extended usinga mixture of individually labeled dideoxynucleoside triphosphates. Thelabel on the extended base is then determined. Preferably, GBA isperformed using semi-automated ELISA or biochip formats (see, e.g., S.R. Head et al. (1997) Nucleic Acids Res. 25(24):5065-71; T. T. Nikiforovet al. (1994) Nucleic Acids Res. 22(20):4167-75).

In another embodiment of the present invention, oligonucleotides, orlonger fragments derived from at least one B7-related polynucleotidesequence described herein may be used as targets in a microarray (e.g.,biochip) system. The microarray can be used to monitor the expressionlevel of large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations, and polymorphisms.This information may be used to determine gene function, to understandthe genetic basis of a disease, to diagnose disease, and to develop andmonitor the activities of therapeutic or prophylactic agents.Preparation and use of microarrays have been described in WO 95/11995 toChee et al.; D. J. Lockhart et al. (1996) Nature Biotechnology14:1675-1680; M. Schena et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; U.S. Pat. No. 6,015,702 to P. Lal et al.; J. Worley etal. (2000) Microarray Biochip Technology, M. Schena, ed., BiotechniquesBook, Natick, Mass., pp. 65-86; Y. H. Rogers et al. (1999) Anal.Biochem. 266(1):23-30; S. R. Head et al. (1999) Mol. Cell. Probes.13(2):81-7; S. J. Watson et al. (2000) Biol. Psychiatry 48(12):1147-56.

In one application of the present invention, microarrays containingarrays of B7-related polynucleotide sequences can be used to measure theexpression levels of B7-related factors in an individual. In particular,to diagnose an individual with a condition or disease correlated withaltered BSL1, BSL2, and/or BSL3 expression levels, a sample from a humanor animal (containing, e.g., mRNA) can be used as a probe on a biochipcontaining an array of BSL1, BSL2, and/or BSL3 polynucleotides (e.g.,DNA) in decreasing concentrations (e.g., 1 ng, 0.1 ng, 0.01 ng, etc.).The test sample can be compared to samples from diseased and normalsamples. Biochips can also be used to identify BSL1, BSL2, and BSL3mutations or polymorphisms in a population, including but not limitedto, deletions, insertions, and mismatches. For example, mutations can beidentified by: (i) placing B7-related polynucleotides of this inventiononto a biochip; (ii) taking a test sample (containing, e.g., mRNA) andadding the sample to the biochip; (iii) determining if the test sampleshybridize to the B7-related polynucleotides attached to the chip undervarious hybridization conditions (see, e.g., V. R. Chechetkin et al.(2000) J. Biomol. Struct. Dyn. 18(1):83-101). Alternatively microarraysequencing can be performed (see, e.g., E. P. Diamandis (2000) Clin.Chem. 46(10):1523-5).

In another embodiment of this invention, a B7-related nucleic acidsequence, or a complementary sequence, or fragment thereof, can be usedas probes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome, or to artificial chromosomeconstructions (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial PI constructions, or singlechromosome cDNA libraries (see C. M. Price (1993) Blood Rev., 7:127-134and by B. J. Trask (1991) Trends Genet. 7:149-154).

In a further embodiment of the present invention, antibodies whichspecifically bind to a BSL1, BSL2, or BSL3 polypeptide may be used forthe diagnosis of conditions or diseases characterized by underexpressionor overexpression of the BSL1, BSL2, or BSL3 polynucleotide orpolypeptide, or in assays to monitor patients being treated with a BSL1,BSL2, or BSL3 polypeptide or peptide, or a BSL1, BSL2, or BSL3 agonist,antagonist, or inhibitor. The antibodies useful for diagnostic purposesmay be prepared in the same manner as those for use in therapeuticmethods, described herein. Diagnostic assays for a BSL1, BSL2, or BSL3polypeptide include methods that utilize the antibody and a label todetect the protein in biological samples (e.g., human body fluids,cells, tissues, or extracts of cells or tissues). The antibodies may beused with or without modification, and may be labeled by joining them,either covalently or non-covalently, with a reporter molecule. A widevariety of reporter molecules that are known in the art may be used,several of which are described herein.

A number of fluorescent materials are known and can be utilized to labela B7-related polypeptide or antibodies that specifically bind thereto.These include, for example, fluorescein, rhodamine, auramine, Texas Red,AMCA blue and Lucifer Yellow. A particular detecting material isanti-rabbit antibody prepared in goats and conjugated with fluoresceinthrough an isothiocyanate. B7-related polypeptides or antibodies theretocan also be labeled with a radioactive element or with an enzyme. Theradioactive label can be detected by any of the currently availablecounting procedures. Preferred isotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S,³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, and ¹⁸⁶ Re.Enzyme labels are likewise useful, and can be detected by any of thepresently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric, or gasometric techniques. Theenzyme can be conjugated by reaction with bridging molecules such ascarbodiimides, diisocyanates, glutaraldehyde, and the like. Manyenzymes, which can be used in these procedures, are known and can beutilized. Preferred are peroxidase, β-glucuronidase, β-D-glucosidase,β-D-galactosidase, urease, glucose oxidase plus peroxidase, and alkalinephosphatase (see, e.g., U.S. Pat. Nos. 3,654,090; 3,850,752; and4,016,043).

Antibody-based diagnostics and their application are familiar to thoseskilled in the art and may be used in accordance with the presentinvention. As non-limiting examples, “competitive” (U.S. Pat. Nos.3,654,090 and 3,850,752), “sandwich” (U.S. Pat. No. 4,016,043), and“double antibody,” or “DASP” assays may be used. Several proceduresincluding ELISA, RIA, and FACS for measuring B7-related polypeptidelevels are known in the art and provide a basis for diagnosing alteredor abnormal levels of B7-related polypeptide expression. Normal orstandard values for B7-related polypeptide expression are established byincubating biological samples taken from normal subjects, preferablyhuman, with antibody to the B7-related polypeptide under conditionssuitable for complex formation. The amount of standard complex formationmay be quantified by various methods; photometric means are preferred.Levels of the B7-related polypeptide expressed in the subject sample,negative control (normal) sample, and positive control (disease) sampleare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

In another of its aspects, this invention relates to diagnostic kits fordetecting B7-related polynucleotide(s) or polypeptide(s) as it relatesto a disease or susceptibility to a disease, particularly the disordersof the immune system described herein. Such kits comprise one or more ofthe following:

(a) a B7-related polynucleotide, preferably the nucleotide sequence ofBSL1 (SEQ ID NO:1 or 3), BSL2 (SEQ ID NO:6, 10, or 12), or BSL3 (SEQ IDNO:14), or a fragment thereof; or

(b) a nucleotide sequence complementary to that of (a); or

(c) a B7-related polypeptide, preferably the polypeptide of BSL1 (SEQ IDNO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15), or afragment thereof; or

(d) an antibody to a B7-related polypeptide, preferably to thepolypeptide of BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), orBSL3 (SEQ ID NO:15), or an antibody bindable fragment thereof. It willbe appreciated that in any such kits, (a), (b), (c), or (d) may comprisea substantial component and that instructions for use can be included.The kits may also contain peripheral reagents such as buffers,stabilizers, etc.

The present invention also includes a test kit for genetic screeningthat can be utilized to identify mutations in B7-related factors. Byidentifying patients with mutated BSL1, BSL2, and/or BSL2 DNA andcomparing the mutation to a database that contains known mutations inBSL1, BSL2, and BSL3, and a particular condition or disease,identification and/or confirmation of, a particular condition or diseasecan be made. Accordingly, such a kit would comprise a PCR-based testthat would involve transcribing the patients mRNA with a specificprimer, and amplifying the resulting cDNA using another set of primers.The amplified product would be detectable by gel electrophoresis andcould be compared with known standards for BSL1, BSL2, and/or BSL3.Preferably, this kit would utilize a patient's blood, serum, or salivasample, and the DNA would be extracted using standard techniques.Primers flanking a known mutation would then be used to amplify afragment of BSL1, BSL2, and/or BSL3. The amplified piece would then besequenced to determine the presence of a mutation.

Therapeutics

Pharmaceutical Compositions:

The present invention contemplates compositions comprising a B7-relatednucleic acid, polypeptide, antibody, ligand, modulator (e.g., agonist,antagonist, or inhibitor), or fragments or functional variants thereof,and a physiologically acceptable carrier, excipient, or diluent asdescribed in detail herein. The present invention further contemplatespharmaceutical compositions useful in practicing the therapeutic methodsof this invention. Preferably, a pharmaceutical composition includes, inadmixture, a pharmaceutically acceptable excipient (carrier) and one ormore of a B7-related polypeptide, nucleic acid, ligand, modulator,antibody, or fragment or functional equivalent thereof, as describedherein, as an active ingredient. Because B7-related polypeptides orpeptides are naturally occurring cellular components, they may beadministered to an individual's circulatory system with minimal risk ofundesired immunological complications.

The preparation of pharmaceutical compositions that contain biologicalreagents as active ingredients is well understood in the art. Typically,such compositions are prepared as injectables, either as liquidsolutions or suspensions, however, solid forms suitable for solution in,or suspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients that are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH-buffering agents, which enhance the effectivenessof the active ingredient.

Pharmaceutical compositions can be produced and employed in treatmentprotocols according to established methods depending on the disorder ordisease to be treated (see, for example, P. D. Mayne (1996) ClinicalChemistry in Diagnosis and Treatment, 6^(th) ed., Oxford UniversityPress, Oxford, England; Gilman et al., Eds. (1990) Goodman and Gilman's:The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; Aviset al., Eds. (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York, N.Y.; and Lieberman et al., Eds. (1990) PharmaceuticalDosage Forms Disperse Systems, Dekker, New York, N.Y.).

Pharmaceutical compositions may be produced as neutral or salt forms.Salts can be formed with many acids, including, but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic and succinicacids. Compositions can take the form of solutions, suspensions,suppositories, tablets, pills, capsules, sustained release compounds, orpowders. Such formulations can contain 10%-95% (w/w) of the activeingredient, preferably 25%-70% (w/w). If the active compound isadministered by injection, for example, about 1 μg-3 mg and preferablyfrom about 20 μg-500 μg of active compound (e.g., B7-relatedpolypeptide) per dosage unit may be administered. Pharmaceuticalpreparations and compositions can also contain one or morephysiologically acceptable carrier(s), excipient(s), diluent(s),disintegrant(s), lubricant(s), plasticizer(s), filler(s), colorant(s),dosage vehicle(s), absorption enhancer(s), stabilizer(s), orbacteriocide(s). The production and formulation of such compositions andpreparations are carried out by methods known and practiced in the art.

Exemplary formulations are given below:

Intravenous Formulation I: Ingredient mg/ml BSL1, BSL2, or BSL3 MAb 5.0dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1water for injection q.s.a.d. 1.0 ml

Intravenous Formulation II: Ingredient mg/ml BSL1, BSL2, or BSL3 MAb 5.0sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injectionq.s.a.d. 1.0 ml

Intravenous Formulation III Ingredient mg/ml BSL1, BSL2, or BSL3protein, 10.0 Ig-fusion protein, or agonist sodium bisulfite USP 3.2disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml

Intravenous Formulation IV Ingredient mg/ml BSL1, BSL2, or BSL3 protein,10.0 Ig-fusion protein, or agonist dextrose USP 45.0 sodium bisulfiteUSP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml

As used herein, “pg” means picogram, “ng” means nanogram, “μg” meanmicrogram, “mg” means milligram, “μl” mean microliter, “ml” meansmilliliter, and “l” means liter.

Following the preparation of pharmaceutical compositions, they may beplaced in appropriate containers and labeled for the treatment ofindicated conditions. Such labeling can include amount, frequency, andmethod of administration. Preparations may be administered systemicallyby oral or parenteral routes. Non-limiting parenteral routes ofadministration include subcutaneous, intramuscular, intraperitoneal,intravenous, transdermal, inhalation, intranasal, intra-arterial,intrathecal, enteral, sublingual, or rectal.

A therapeutically effective amount of a pharmaceutical compositioncontaining one or more B7-related polypeptides, fusion proteins, peptidefragments, or antibodies that specifically react with these componentsis an amount sufficient to reduce, ameliorate, or eliminate a disease ordisorder related to altered activation levels of immune or inflammatoryresponse cells, such as T-cells. An effective amount can be introducedin one administration or over repeated administrations to an individualbeing treated. Therapeutic administration can be followed byprophylactic administration, after treatment of the disease. Aprophylactically effective amount is an amount effective to preventdisease and will depend upon the specific illness and subject. Thetherapeutically effective dose may be estimated initially, for example,either in cell culture assays or in animal models, usually mice, rats,rabbits, dogs, sheep, goats, pigs, or non-human primates. The animalmodel may also be used to determine the maximum tolerated dose andappropriate route of administration. Such information can then be usedto determine useful doses and routes for administration in humans.

Administration of the therapeutic compositions of the present inventionto a subject can be carried out using known procedures, at dosages andfor periods of time effective to achieve the desired result. Forexample, a therapeutically active amount B7-related polypeptides, fusionproteins, peptides, or antibodies that react with these components mayvary according to factors such as the age, sex, and weight of theindividual, and the ability of the treatment to elicit a desiredresponse in the individual. Dosages may be adjusted to provide theoptimum therapeutic response. For example, several sequential doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Gene Transfer Therapy:

In addition, host cells that are genetically engineered to carry thegene encoding a B7-related polypeptide, fusion protein, or peptidefragment comprising a fragment of the BSL1 (SEQ ID NO:2), BSL2 (SEQ IDNO:7, 11, or 13), or BSL3 (SEQ ID NO:15) polypeptide sequence, can beintroduced into an individual in need of immunomodulation. Followingexpression and production of the B7-related polypeptide or peptide bythe host cell, the so-produced B7-related polypeptide, fusion protein,or peptide can act to bind CD28 and/or CD28-related ligand(s) tomodulate the activation of immune or inflammatory response cells (e.g.,T-cells) in the recipient. Host cells may be genetically engineered by avariety of molecular techniques and methods known to those having skillin the art, for example, transfection, infection, or transduction.Transduction as used herein commonly refers to cells that have beengenetically engineered to contain a foreign or heterologous gene via theintroduction of a viral or non-viral vector into the cells. Transfectionmore commonly refers to cells that have been genetically engineered tocontain a foreign gene harbored in a plasmid, or non-viral vector. Hostcells can be transfected or transduced by different vectors and thus canserve as gene delivery vehicles to transfer the expressed products intomuscle.

Although viral vectors are preferred for gene transfer therapies, cellscan be genetically engineered to contain nucleic acid sequences encodingthe desired gene product(s) by various methods known in the art. Forexample, cells can be genetically engineered by fusion, transfection,lipofection mediated by the use of liposomes, electroporation,precipitation with DEAE-Dextran or calcium phosphate, particlebombardment (biolistics) with nucleic acid-coated particles (e.g., goldparticles), microinjection, or genetically engineered microorganisms (K.Yazawa et al. (2000) Cancer Gene Ther. 7:269-274). Vectors forintroducing heterologous (i.e., foreign) nucleic acid (DNA or RNA) intomuscle cells for the expression of active bioactive products are wellknown in the art. Such vectors possess a promoter sequence, preferably apromoter that is cell-specific and placed upstream of the sequence to beexpressed. The vectors may also contain, optionally, one or moreexpressible marker genes for expression as an indication of successfultransfection and expression of the nucleic acid sequences contained inthe vector. In addition, vectors can be optimized to minimize undesiredimmunogenicity and maximize long-term expression of the desired geneproduct(s) (see Nabel (1999) Proc. Natl. Acad. Sci. USA 96:324-326).Moreover, vectors can be chosen based on cell-type that is targeted fortreatment. For example, vectors for the treatment of tumor or cancercells have been described (P. L. Hallenbeck et al. (1999) Hum. GeneTher. 10:1721-1733; T. Shibata et al. (2000) Gene Ther. 7:493-498; M.Puhlmann et al. (2000) Cancer Gene Ther. 7:66-73; N. Krauzewicz et al.(2000) Adv. Exp. Med. Biol. 465:73-82).

Illustrative examples of vehicles or vector constructs for transfectionor infection of the host cells include replication-defective viralvectors, DNA virus or RNA virus (retrovirus) vectors, such asadenovirus, herpes simplex virus and adeno-associated viral vectors.Adeno-associated virus vectors are single stranded and allow theefficient delivery of multiple copies of nucleic acid to the cell'snucleus. Preferred are adenovirus vectors. The vectors will normally besubstantially free of any prokaryotic DNA and may comprise a number ofdifferent functional nucleic acid sequences. An example of suchfunctional sequences may be a DNA region comprising transcriptional andtranslational initiation and termination regulatory sequences, includingpromoters (e.g., strong promoters, inducible promoters, and the like)and enhancers which are active in the host cells. Also included as partof the functional sequences is an open reading frame (polynucleotidesequence) encoding a protein of interest. Flanking sequences may also beincluded for site-directed integration. In some situations, the5′-flanking sequence will allow homologous recombination, thus changingthe nature of the transcriptional initiation region, so as to providefor inducible or noninducible transcription to increase or decrease thelevel of transcription, as an example.

In general, the encoded and expressed B7-related factor may beintracellular, i.e., retained in the cytoplasm, nucleus, or an organelleof a cell, or may be secreted by the cell. For secretion, the naturalsignal sequence present in the B7-related structural gene may beretained. When the polypeptide or peptide is a fragment of a B7-relatedfactor that is larger, a signal sequence may be provided so that, uponsecretion and processing at the processing site, the desired proteinwill have the natural sequence. Specific examples of coding sequences ofinterest for use in accordance with the present invention include theBSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ IDNO:15) polypeptide coding sequences. As previously mentioned, a markermay be present for selection of cells containing the vector construct.The marker may be an inducible or non-inducible gene and will generallyallow for positive selection under induction, or without induction,respectively. Examples of marker genes include neomycin, dihydrofolatereductase, glutamine synthetase, and the like.

The vector employed will generally also include an origin of replicationand other genes that are necessary for replication in the host cells, asroutinely employed by those having skill in the art. As an example, thereplication system comprising the origin of replication and any proteinsassociated with replication encoded by a particular virus may beincluded as part of the construct. The replication system must beselected so that the genes encoding products necessary for replicationdo not ultimately transform the cells. Such replication systems arerepresented by replication-defective adenovirus (see G. Acsadi et al.(1994) Hum. Mol. Genet. 3:579-584) and by Epstein-Barr virus. Examplesof replication defective vectors, particularly, retroviral vectors thatare replication defective, are BAG, (see Price et al. (1987) Proc. Natl.Acad. Sci. USA, 84:156; Sanes et al. (1986) EMBO J., 5:3133). It will beunderstood that the final gene construct may contain one or more genesof interest, for example, a gene encoding a bioactive metabolicmolecule. In addition, cDNA, synthetically produced DNA or chromosomalDNA may be employed utilizing methods and protocols known and practicedby those having skill in the art.

According to one approach for gene therapy, a vector encoding aB7-related factor is directly injected into the recipient cells (in vivogene therapy). Alternatively, cells from the intended recipients areexplanted, genetically modified to encode a B7-related factor, andreimplanted into the donor (ex vivo gene therapy). An ex vivo approachprovides the advantage of efficient viral gene transfer, which issuperior to in vivo gene transfer approaches. In accordance with ex vivogene therapy, the host cells are first infected with engineered viralvectors containing at least one B7-related gene encoding a B7-relatedgene product, suspended in a physiologically acceptable carrier orexcipient such as saline or phosphate buffered saline, and the like, andthen administered to the host. The desired gene product is expressed bythe injected cells, which thus introduce the gene product into the host.The introduced gene products can thereby be utilized to treat orameliorate a disorder that is related to altered levels of theactivation of immune or inflammatory response cells (e.g., T-cells).

Methods of Immunomodulation:

In accordance with the present invention, the BSL1, BSL2, and BSL3nucleic acid and polypeptide sequences can be used in the development oftherapeutic reagents having the ability to either up-regulate (amplify)or down-regulate (suppress) immune responses (e.g., T-cell activation).In particular, B7-related polypeptides may interact with CD28 andthereby up-regulate immune cell activity. Alternatively, B7-relatedpolypeptides may interact with CTLA-4 and thereby down-regulate immunecell activity. For example, soluble, dimeric forms of the B7-relatedpolypeptides that bind to the CD28 and/or CD28-related ligand(s) butfail to provide a costimulatory signal to T-cells, can be used to blockT-cell activation, and thereby provide a specific means by which toinduce tolerance in a subject. Similarly, antibodies directed againstone or more B7-related factors can be used to block the interactionbetween the B7-related factors and their cognate ligand(s), therebypreventing the activation of immune or inflammatory response cells(e.g., T-cells). In addition, fusion proteins comprising at least afragment of a B7-related factor fused to at least the Fc domain of anIgG molecule can be used to up- or down-regulate cells expressing thecognate ligand(s) of the B7-related factor (e.g., T-cells). Furthermore,antisense or triplex oligonucleotides that bind to the nucleotidesequence of one or more B7-related factors can be used to decrease theexpression these factors. In contrast, cell surface, multivalent formsof B7-related factors that bind to CD28 and/or CD28-related ligand(s)and provide a costimulatory signal to immune or inflammatory responsecells, such as T-cells, can be used to increase the activation of thesecells. It is also possible to utilize more than one B7-related factor,fusion protein, antibody, or therapeutically active fragments thereof,in order to up-regulate or down-regulate the activity of immune orinflammatory cells (e.g., T-cells) in an animal or human subject.

More specifically, given the structure and function of the B7-relatedfactors disclosed herein, it is possible to up-regulate or down-regulatethe function of a B7-related factor in a number of ways. Down-regulatingor preventing one or more B7-related factor functions (i.e., preventinghigh level lymphokine synthesis by activated T-cells) should be usefulin treating autoimmune diseases, such as rheumatoid arthritis, multiplesclerosis, Lupus erythematosus, Hashimoto's thyroiditis, primarymixedema, Graves' disease, pernicious anemia, autoimmune atrophicgastritis, insulin dependent diabetes mellitus, good pasture's syndrome,myasthenia gravis, pemphigus, Crohn's disease, sympathetic opthalmia,autoimmune uveitis, autoimmune hemolytic anemis, idiopathicthrombocytopenia, primary biliary cirrhosis, ulcerative colitis,Sjogren's syndrome, polymyositis and mixed connective tissue disease.B7-related factors may also be down-regulated for the treatment ofinflammation related to psoriasis, chronic obstructive pulmonarydisease, asthma, and atherosclerosis. In addition, B7-related factorsmay be down-regulated for the treatment of tissue, bone marrow, andorgan transplantation, and graft versus host disease. For example,blockage of T-cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated by its recognition as foreign material,followed by an immune reaction that destroys the transplant. TheB7-related molecules of the present invention can also be used to treator prevent cancers as described in detail below.

The B7-related nucleic acid molecules provided by the present inventioncan be used to design therapeutics to block the function of one or moreB7-related factors. In particular, antisense or triplex oligonucleotidescan be administered to prevent the expression of the BSL1, BSL2, and/orBSL3 factors. For example, an oligonucleotide (e.g., DNAoligonucleotide) that hybridizes to the BSL1, BSL2, and/or BSL3 mRNA canbe used to target the mRNA for RnaseH digestion. Alternatively, anoligonucleotide that hybridizes to the translation initiation site ofthe BSL1 (SEQ ID NO:1 or 3), BSL2 (SEQ ID NO:6, 10, or 12), or BSL3 (SEQID NO:14) mRNA be used to prevent translation of the mRNA. In anotherapproach, oligonucleotides that bind to the double-stranded DNA of theBSL1, BSL2, and/or BSL3 gene(s) can be administered. Sucholigonucleotides can form a triplex construct and prevent the unwindingand transcription of the DNA encoding the targeted B7-related factor. Inall cases, the appropriate oligonucleotide can be synthesized,formulated as a pharmaceutical composition, and administered to asubject. The synthesis and utilization of antisense and triplexoligonucleotides have been previously described (e.g., H. Simon et al.(1999) Antisense Nucleic Acid Drug Dev. 9:527-31; F. X. Barre et al.(2000) Proc. Natl. Acad. Sci. USA 97:3084-3088; R. Elez et al. (2000)Biochem. Biophys. Res. Commun. 269:352-6; E. R. Sauter et al. (2000)Clin. Cancer Res. 6:654-60).

In the context of this invention, antisense oligonucleotides arenaturally-occurring oligonucleotide species or synthetic species formedfrom naturally-occurring subunits or their close homologs. Antisenseoligonucleotides may also include moieties that function similarly tooligonucleotides, but have non-naturally-occurring portions. Thus,antisense oligonucleotides may have altered sugar moieties orinter-sugar linkages. Exemplary among these are phosphorothioate andother sulfur containing species which are known in the art.

In preferred embodiments, at least one of the phosphodiester bonds ofthe antisense oligonucleotide has been substituted with a structure thatfunctions to enhance the ability of the compositions to penetrate intothe region of cells where the RNA whose activity is to be modulated islocated. It is preferred that such substitutions comprisephosphorothioate bonds, methyl phosphonate bonds, or short chain alkylor cycloalkyl structures. In accordance with other preferredembodiments, the phosphodiester bonds are substituted with structureswhich are, at once, substantially non-ionic and non-chiral, or withstructures which are chiral and enantiomerically specific. Persons ofordinary skill in the art will be able to select other linkages for usein the practice of the invention.

Antisense oligonucleotides may also include species that include atleast some modified base forms. Thus, purines and pyrimidines other thanthose normally found in nature may be so employed. Similarly,modifications on the furanosyl portions of the nucleotide subunits mayalso be effected, as long as the essential tenets of this invention areadhered to. Examples of such modifications are 2′-O-alkyl- and2′-halogen-substituted nucleotides. Some non-limiting examples ofmodifications at the 2′ position of sugar moieties which are useful inthe present invention include OH, SH, SCH₃, F, OCH₃, OCN, O(CH₂)_(n) NH₂and O(CH₂)_(n) CH₃, where n is from 1 to about 10. Such antisenseoligonucleotides are functionally interchangeable with naturaloligonucleotides or synthesized oligonucleotides, which have one or moredifferences from the natural structure. All such analogs arecomprehended by this invention so long as they function effectively tohybridize with BSL1, BSL2, or BSL3 DNA or RNA to inhibit the functionthereof.

For antisense therapeutics, the oligonucleotides in accordance with thisinvention preferably comprise from about 3 to about 50 subunits. It ismore preferred that such oligonucleotides and analogs comprise fromabout 8 to about 25 subunits and still more preferred to have from about12 to about 20 subunits. As defined herein, a “subunit” is a base andsugar combination suitably bound to adjacent subunits throughphosphodiester or other bonds.

Antisense oligonulcleotides can be produced by standard techniques (see,e.g., Shewmaker et al., U.S. Pat. No. 5,107,065). The oligonucleotidesused in accordance with this invention may be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is available from several vendors,including PE Applied Biosystems (Foster City, Calif.). Any other meansfor such synthesis may also be employed, however, the actual synthesisof the oligonucleotides is well within the abilities of thepractitioner. It is also will known to prepare other oligonucleotidesuch as phosphorothioates and alkylated derivatives.

The oligonucleotides of this invention are designed to be hybridizablewith BSL1, BSL2, or BSL3 RNA (e.g., mRNA) or DNA. For example, anoligonucleotide (e.g., DNA oligonucleotide) that hybridizes toB7-related mRNA can be used to target the mRNA for RnaseH digestion.Alternatively, an oligonucleotide that hybridizes to the translationinitiation site of B7-related mRNA can be used to prevent translation ofthe mRNA. In another approach, oligonucleotides that bind to thedouble-stranded DNA of BSL1, BSL2, or BSL3 can be administered. Sucholigonucleotides can form a triplex construct and inhibit thetranscription of the DNA encoding BSL1, BSL2, or BSL3 polypeptides.Triple helix pairing prevents the double helix from opening sufficientlyto allow the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described (see, e.g., J. E. Gee et al. (1994) Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).

As non-limiting examples, antisense oligonucleotides may be targeted tohybridize to the following regions: mRNA cap region; translationinitiation site; translational termination site; transcriptioninitiation site; transcription termination site; polyadenylation signal;3′ untranslated region; 5′ untranslated region; 5′ coding region; midcoding region; and 3′ coding region. Preferably, the complementaryoligonucleotide is designed to hybridize to the most unique 5′ sequencein BSL1, BSL2, or BSL3, including any of about 15-35 nucleotidesspanning the 5′ coding sequence. Appropriate oligonucleotides can bedesigned using OLIGO software (Molecular Biology Insights, Inc.,Cascade, Colo.).

In accordance with the present invention, the antisense oligonucleotidecan be synthesized, formulated as a pharmaceutical composition, andadministered to a subject. The synthesis and utilization of antisenseand triplex oligonucleotides have been previously described (e.g., H.Simon et al. (1999) Antisense Nucleic Acid Drug Dev. 9:527-31; F. X.Barre et al. (2000) Proc. Natl. Acad. Sci. USA 97:3084-3088; R. Elez etal. (2000) Biochem. Biophys. Res. Commun. 269:352-6; E. R. Sauter et al.(2000) Clin. Cancer Res. 6:654-60). Alternatively, expression vectorsderived from retroviruses, adenovirus, herpes or vaccinia viruses, orfrom various bacterial plasmids may be used for delivery of nucleotidesequences to the targeted organ, tissue or cell population. Methodswhich are well known to those skilled in the art can be used toconstruct recombinant vectors which will express nucleic acid sequencethat is complementary to the nucleic acid sequence encoding a BSL1,BSL2, or BSL3 polypeptide. These techniques are described both inSambrook et al. (1989) and in Ausubel et al. (1992). For example, BSL1,BSL2, or BSL3 expression can be inhibited by transforming a cell ortissue with an expression vector that expresses high levels ofuntranslatable sense or antisense sequences. Even in the absence ofintegration into the DNA, such vectors may continue to transcribe RNAmolecules until they are disabled by endogenous nucleases. Transientexpression may last for a month or more with a non-replicating vector,and even longer if appropriate replication elements included in thevector system.

Various assays may be used to test the ability of specific antisenseoligonucleotides to inhibit BSL1, BSL2, or BSL3 expression. For example,mRNA levels can be assessed northern blot analysis (Sambrook et al.(1989); Ausubel et al. (1992); J. C. Alwine et al. (1977) Proc. Natl.Acad. Sci. USA 74:5350-5354; I. M. Bird (1998) Methods Mol. Biol.105:325-36), quantitative or semi-quantitative RT-PCR analysis (see,e.g., W. M. Freeman et al. (1999) Biotechniques 26:112-122; Ren et al.(1998) Mol. Brain Res. 59:256-63; J. M. Cale et al. (1998), Methods Mol.Biol. 105:351-71), or in situ hybridization (reviewed by A. K. Reap(1998) Mutat. Res. 400:287-298). Alternatively, antisenseoligonucleotides may be assessed by measuring levels of BSL1, BSL2, orBSL3 polypeptide, e.g., by western blot analysis, indirectimmunofluorescence, immunoprecipitation techniques (see, e.g., J. M.Walker (1998) Protein Protocols on CD-ROM, Humana Press, Totowa, N.J.).

The B7-related polypeptide sequences provided by the present inventionmay also be useful in the design of therapeutic agents to block orenhance the activity of immune response cells (e.g., T-cells). Forexample, a fusion protein comprising the soluble portion of a B7-relatedpolypeptide conjugated with the Fc domain of human IgG can beconstructed by standard recombinant techniques, described above. TheBSL1-Ig, BSL2-Ig, and/or BSL3-Ig, fusion proteins can be prepared as apharmaceutical composition and administered to a subject. The BSL1-Ig,BSL2-Ig, and/or BSL3-Ig fusion proteins can be used to target specificT-cells for destruction, thereby reducing overall T-cell activation.Such treatment methods can be modeled on animal experiments, whichutilize CTLA-4-Ig to prevent cardiac allograft rejection (Turka et al.,supra). It will be understood by a person skilled in the art that suchmethods may be adapted for use in humans, and for use with otherconditions, including various transplants and autoimmune diseases.Alternatively, the BSL1-Ig, BSL2-Ig, and/or BSL3-Ig fusion proteins canbe used to enhance T-cell activation. For example, BSL2-Ig and BSL3-Igfusion proteins can be used as co-stimulatory molecules as disclosed indetail herein.

As an alternative approach, antibodies that specifically react withB7-related polypeptides or peptides can be used to block the activity ofimmune or inflammatory response cells (e.g., T-cells). Antibodies orrelated antibody fragments that bind to peptides or polypeptidescomprising the BSL1 (SEQ ID NO:2), BSL2 (SEQ ID NO:7, 11, or 13), orBSL3 (SEQ ID NO:15) sequences can be formulated as pharmaceuticalcompositions and administered alone or in combination to a subject. Suchantibodies can then inhibit the interaction of the B7-relatedpolypeptides with CD28 and/or CD28-related ligands, and thereby preventT-cell activation. Treatments utilizing antibodies directed againstB7-related factors may be modeled on animal experiments, which useantibodies against CD28, B7-1, or B7-2 (D. J. Lenshow et al. (1995)Transplantation 60:1171-1178; Y. Seko et al. (1998) Circ. Res.83:463-469; A. Haczku et al. (1999) Am. J. Respir. Crit. Care Med.159:1638-1643). One skilled in the art may adapt such methods for use inhumans, and for use with various conditions involving inflammation ortransplantation.

It is noted that antibody-based therapeutics produced from non-humansources can cause an undesired immune response in human subjects. Tominimize this problem, chimeric antibody derivatives can be produced.Chimeric antibodies combine a non-human animal variable region with ahuman constant region. Chimeric antibodies can be constructed accordingto methods known in the art (see Morrison et al. (1985) Proc. Natl.Acad. Sci. USA 81:6851; Takeda et al. (1985) Nature 314:452; U.S. Pat.No. 4,816,567 of Cabilly et al.; U.S. Pat. No. 4,816,397 of Boss et al.;European Patent Publication EP 171496; EP 0173494; United Kingdom PatentGB 2177096B). In addition, antibodies can be further “humanized” by anyof the techniques known in the art, (e.g., Teng et al. (1983) Proc.Natl. Acad. Sci. USA 80:7308-7312; Kozbor et al. (1983) Immunology Today4: 7279; Olsson et al. (1982) Meth. Enzymol. 92:3-16; InternationalPatent Application No. WO 92/06193; EP 0239400). Humanized antibodiescan be also be obtained from commercial sources (e.g., Scotgen Limited,Middlesex, Great Britain). Immunotherapy with a humanized antibody mayresult in increased long-term effectiveness for the treatment of chronicdisease situations or situations requiring repeated antibody treatments.

In yet another approach, an isolated ligand of a B7-related factor canbe used to down-regulate the activity of immune or inflammatory responsecells (e.g., T-cells). For example, a soluble fusion protein comprisinga B7-related factor ligand can be produced, isolated, and used toproduce a pharmaceutical composition in accordance with the methodsdescribed in detail herein. This pharmaceutical composition can then beadministered to a subject to bind to one or more endogenous B7-relatedfactor(s) and block the activation of immune or inflammatory responsecells (e.g., T-cells) as previously described.

Up-regulation of a B7-related factor function may also be useful intherapy. Because viral infections are cleared primarily by cytotoxicT-cells, an increase in cytotoxic activity would be therapeuticallyuseful in situations where more rapid or thorough clearance of aninfective viral agent would be beneficial to an animal or human subject.Notably, B7-1 acts to increase the cytotoxicity of T-cells thoughinteractions with its cognate ligand(s). Thus, soluble active forms ofB7-related polypeptides can be administered for the treatment of localor systemic viral infections, such as immunodeficiency (e.g., HIV),papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza(e.g., human influenza virus A), and common cold (e.g., humanrhinovirus) viral infections. For example, pharmaceutical formulationsof active multivalent B7-related factors can be administered topicallyto treat viral skin diseases such as herpes lesions or shingles, orgenital warts. Alternatively, pharmaceutical compositions of active,multivalent B7-related factors can be administered systemically to treatsystemic viral diseases such as AIDS, influenza, the common cold, orencephalitis.

In addition, modulation of B7-related factor function may be useful inthe induction of tumor immunity. For example, tumor cells (e.g.,sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, or carcinomacells) can be genetically engineered to carry a nucleic acid encoding atleast a fragment of at least one B7-related factor, such as BSL1 (SEQ IDNO:2), BSL2 (SEQ ID NO:7, 11, or 13), or BSL3 (SEQ ID NO:15), and thenadministered to a subject to traverse tumor-specific tolerance in thesubject. Notably, ectopic expression of B7-1 in B7 negative murine tumorcells has been shown to induce T-cell mediated specific immunityaccompanied by tumor rejection and prolonged protection to tumorchallenge in mice (L. Chen et al., supra; S. Townsend et al., supra; S.Baskar et al., supra). Tumor or cancer cell gene therapy treatmentsutilizing B7-related factors may be modeled on animal experiments (seeK. Dunussi-Joannopoulos et al. (1997) J. Pediatr. Hematol. Oncol.19:356-340; K. Hiroishi et al. (1999) Gene Ther. 6:1988-1994; B. K.Martin et al. (1999) J. immunol. 162:6663-6670; M. Kuiper et al. (2000)Adv. Exp. Med. Biol. 465:381-390), or human phase I trial experiments(H. L. Kaufman et al. (2000) Hum. Gene Ther. 11:1065-1082), which useB7-1 or B7-2 for gene transfer therapy. It will be understood that suchmethods may be adapted for use with various tumor or cancer cells.Additionally, tumor immunity may be achieved by administration of aB7-related fusion protein that directly stimulates the immune cells (seee.g., International Patent Application No. WO 01/21796 to V. Ling etal.).

Pharmacogenetics:

The B7-related polypeptides and polynucleotides of the present inventionare also useful in pharmacogenetic analysis, i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a therapeutic composition or drug. See, e.g., Eichelbaum, M.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985, and Linder, M.W. (1997) Clin. Chem. 43(2):254-266. The genotype of the individual candetermine the way a therapeutic acts on the body or the way the bodymetabolizes the therapeutic. Further, the activity of drug metabolizingenzymes affects both the intensity and duration of therapeutic activity.Differences in the activity or metabolism of therapeutics can lead tosevere toxicity or therapeutic failure. Accordingly, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenetic studies in determining whether to administer aB7-related polypeptide, polynucleotide, functional equivalent, fragment,or modulator, as well as tailoring the dosage and/or therapeutic orprophylactic treatment regimen with a B7-related polypeptide,polynucleotide, functional equivalent, fragment, or modulator.

In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions can be due to a single factor thatalters the way the drug act on the body (altered drug action), or afactor that alters the way the body metabolizes the drug (altered drugmetabolism). These conditions can occur either as rare genetic defectsor as naturally occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy which results in haemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

The discovery of genetic polymorphisms of drug metabolizing enzymes(e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6and CYP2C19) has provided an explanation as to why some patients do notobtain the expected drug effects or show exaggerated drug response andserious toxicity after taking the standard and safe dose of a drug.These polymorphisms are expressed in two phenotypes in the population,the extensive metabolizer (EM) and poor metabolizer (PM). The prevalenceof PM is different among different populations. The gene coding forCYP2D6 is highly polymorphic and several mutations have been identifiedin PM, which all lead to the absence of functional CYP2D6. Poormetabolizers quite frequently experience exaggerated drug response andside effects when they receive standard doses. If a metabolite is theactive therapeutic moiety, PM show no therapeutic response. This hasbeen demonstrated for the analgesic effect of codeine mediated by itsCYP2D6-formed metabolite morphine. At the other extreme, ultra-rapidmetabolizers fail to respond to standard doses. Recent studies havedetermined that ultra-rapid metabolism is attributable to CYP2D6 geneamplification.

By analogy, genetic polymorphism or mutation may lead to allelicvariants of BSL1, BSL2, and/or BSL3 in the population which havedifferent levels of activity. The BSL1, BSL2, and/or BSL3 polypeptidesor polynucleotides thereby allow a clinician to ascertain a geneticpredisposition that can affect treatment modality. Thus, in a BSL-basedtreatment, polymorphism or mutation may give rise to individuals thatare more or less responsive to treatment. Accordingly, dosage wouldnecessarily be modified to maximize the therapeutic effect within agiven population containing the polymorphism. As an alternative togenotyping, specific polymorphic polypeptides or polynucleotides can beidentified.

To identify genes that predict drug response, several pharmacogeneticmethods can be used. One pharmacogenomics approach, “genome-wideassociation”, relies primarily on a high-resolution map of the humangenome. This high-resolution map shows previously identifiedgene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants). A high-resolution genetic mapcan then be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, a high-resolution map can be generatedfrom a combination of some 10 million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Inthis way, treatment regimens can be tailored to groups of geneticallysimilar individuals, taking into account traits that may be common amongsuch genetically similar individuals. See, e.g., D. R. Pfost et al.(2000) Trends Biotechnol. 18(8):334-8.

As another example, the “candidate gene approach”, can be used.According to this method, if a gene that encodes a drug target is known,all common variants of that gene can be fairly easily identified in thepopulation and it can be determined if having one version of the geneversus another is associated with a particular drug response.

As yet another example, a “gene expression profiling approach”, can beused. This method involves testing the gene expression of an animaltreated with a drug (e.g., a B7-related polypeptide, polynucleotide,functional equivalent, fragment, or modulator) to determine whether genepathways related to toxicity have been turned on.

Information obtained from one of the pharmacogenetics approachesdescribed herein can be used to determine appropriate dosage andtreatment regimens for prophylactic or therapeutic treatment anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with aB7-related polypeptide, polynucleotide, functional equivalent, fragment,or modulator.

B7-related polypeptides or polynucleotides are also useful formonitoring therapeutic effects during clinical trials and othertreatment. Thus, the therapeutic effectiveness of an agent that isdesigned to increase or decrease gene expression, polypeptide levels, oractivity can be monitored over the course of treatment using theB7-related polypeptides or polynucleotides. For example, monitoring canbe performed by: (i) obtaining a pre-administration sample from asubject prior to administration of the agent; (ii) detecting the levelof expression or activity of the polypeptide in the pre-administrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of thepolypeptide in the post-administration samples; (v) comparing the levelof expression or activity of the polypeptide in the pre-administrationsample with the polypeptide in the post-administration sample orsamples; and (vi) increasing or decreasing the administration of theagent to the subject accordingly.

Animal Models

B7-related polynucleotides can be used to generate genetically alterednon-human animals or human cell lines. Any non-human animal can be used;however typical animals are rodents, such as mice, rats, or guinea pigs.Genetically engineered animals or cell lines can carry a gene that hasbeen altered to contain deletions, substitutions, insertions, ormodifications of the polynucleotide sequence (e.g., exon sequence). Suchalterations may render the gene nonfunctional, (i.e., a null mutation)producing a “knockout” animal or cell line. In addition, geneticallyengineered animals can carry one or more exogenous or non-naturallyoccurring genes, e.g., “transgenes” or “orthologs”, that are derivedfrom different organisms (e.g., humans), or produced by synthetic orrecombinant methods. Genetically altered animals or cell lines can beused to study BSL1, BSL2, or BSL3 function, regulation, and to developtreatments for BSL1-, BSL2-, or BSL3-related diseases. In particular,knockout animals and cell lines can be used to establish animal modelsand in vitro models for analysis of BSL1-, BSL2-, or BSL3-relateddiseases. In addition, transgenic animals expressing human BSL1, BSL2,or BSL3 can be used in drug discovery efforts.

A “transgenic animal” is any animal containing one or more cells bearinggenetic information altered or received, directly or indirectly, bydeliberate genetic manipulation at a subcellular level, such as bytargeted recombination or microinjection or infection with recombinantvirus. The term “transgenic animal” is not intended to encompassclassical cross-breeding or in vitro fertilization, but rather is meantto encompass animals in which one or more cells are altered by, orreceive, a recombinant DNA molecule. This recombinant DNA molecule maybe specifically targeted to a defined genetic locus, may be randomlyintegrated within a chromosome, or it may be extrachromosomallyreplicating DNA.

As used herein, the term “ortholog” denotes a gene or polypeptideobtained from one species that has homology to an analogous gene orpolypeptide from a different species. For example, the human BSL3 (SEQID NO:15) and mouse AF142780 polypeptides are orthologs.

Transgenic animals can be selected after treatment of germline cells orzygotes. For example, expression of an exogenous BSL1, BSL2, or BSL3gene or a variant can be achieved by operably linking the gene to apromoter and optionally an enhancer, and then microinjecting theconstruct into a zygote (see, e.g., Hogan et al. (1994) Manipulating theMouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.). Such treatments include insertion of the exogenousgene and disrupted homologous genes. Alternatively, the gene(s) of theanimals may be disrupted by insertion or deletion mutation of othergenetic alterations using conventional techniques (see, e.g., Capecchi,(1989) Science, 244:1288; Valancuis et al. (1991) Mol. Cell. Biol.,11:1402; Hasty et al. (1991) Nature, 350:243; Shinkai et al. (1992)Cell, 68:855; Mombaerts et al. (1992) Cell, 68:869; Philpott et al.(1992) Science, 256:1448; Snouwaert et al. (1992) Science, 257:1083;Donehower et al. (1992) Nature, 356:215).

In one aspect of the invention, BSL1, BSL2, or BSL3 knockout mice can beproduced in accordance with well-known methods (see, e.g., M. R.Capecchi, (1989) Science, 244:1288-1292; P. Li et al. (1995) Cell80:401-411; L. A. Galli-Taliadoros et al. (1995) J. Immunol. Methods181(1):1-15; C. H. Westphal et al. (1997) Curr. Biol. 7(7):530-3; S. S.Cheah et al. (2000) Methods Mol. Biol. 136:455-63). The human BSL1,BSL2, and BSL3 clones can be used isolate murine homologs. A murinehomologs can then be used to prepare a murine BSL1, BSL2, or BSL3targeting construct that can disrupt BSL1, BSL2, or BSL3 in the mouse byhomologous recombination at the corresponding chromosomal locus. Thetargeting construct can comprise a disrupted or deleted murine BSL1,BSL2, or BSL3 sequence that inserts in place of the functioning fragmentof the native mouse gene. For example, the construct can contain aninsertion in the murine BSL1, BSL2, or BSL3 protein-coding region.

Preferably, the targeting construct contains markers for both positiveand negative selection. The positive selection marker allows theselective elimination of cells that lack the marker, while the negativeselection marker allows the elimination of cells that carry the marker.In particular, the positive selectable marker can be an antibioticresistance gene, such as the neomycin resistance gene, which can beplaced within the coding sequence of murine BSL1, BSL2, or BSL3 torender it non-functional, while at the same time rendering the constructselectable. The herpes simplex virus thymidine kinase (HSV tk) gene isan example of a negative selectable marker that can be used as a secondmarker to eliminate cells that carry it. Cells with the HSV tk gene areselectively killed in the presence of gangcyclovir. As an example, apositive selection marker can be positioned on a targeting constructwithin the region of the construct that integrates at the BSL1, BSL2, orBSL3 locus. The negative selection marker can be positioned on thetargeting construct outside the region that integrates at the BSL1,BSL2, or BSL3 locus. Thus, if the entire construct is present in thecell, both positive and negative selection markers will be present. Ifthe construct has integrated into the genome, the positive selectionmarker will be present, but the negative selection marker will be lost.

The targeting construct can be employed, for example, in embryonal stemcell (ES). ES cells may be obtained from pre-implantation embryoscultured in vitro (M. J. Evans et al. (1981) Nature 292:154-156; M. O.Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) Proc.Natl. Acad. Sci. USA 83:9065-9069; Robertson et al. (1986) Nature322:445-448; S. A. Wood et al. (1993) Proc. Natl. Acad. Sci. USA90:4582-4584). Targeting constructs can be efficiently introduced intothe ES cells by standard techniques such as DNA transfection or byretrovirus-mediated transduction. Following this, the transformed EScells can be combined with blastocysts from a non-human animal. Theintroduced ES cells colonize the embryo and contribute to the germ lineof the resulting chimeric animal (R. Jaenisch, (1988) Science240:1468-1474). The use of gene-targeted ES cells in the generation ofgene-targeted transgenic mice has been previously described (Thomas etal. (1987) Cell 51:503-512) and is reviewed elsewhere (Frohman et al.(1989) Cell 56:145-147; Capecchi, 1989, Trends in Genet. 5:70-76;Baribault et al. (1989) Mol. Biol. Med. 6:481-492; Wagner, (1990) EMBOJ. 9:3025-3032; Bradley et al. (1992) Bio/Technology 10: 534-539).

Several methods can be used to select homologously recombined murine EScells. One method employs PCR to screen pools of transformant cells forhomologous insertion, followed by screening individual clones (Kim etal. (1988) Nucleic Acids Res. 16:8887-8903; Kim et al. (1991) Gene103:227-233). Another method employs a marker gene is constructed whichwill only be active if homologous insertion occurs, allowing theserecombinants to be selected directly (Sedivy et al. (1989) Proc. Natl.Acad. Sci. USA 86:227-231). For example, the positive-negative selection(PNS) method can be used as described above (see, e.g., Mansour et al.(1988) Nature 336:348-352; Capecchi, 1989, Science 244:1288-1292;Capecchi, (1989) Trends in Genet. 5:70-76). In particular, the PNSmethod is useful for targeting genes that are expressed at low levels.

The absence of functional BSL1, BSL2, or BSL3 in the knockout mice canbe confirmed, for example, by RNA analysis, protein expression analysis,and functional studies. For RNA analysis, RNA samples are prepared fromdifferent organs of the knockout mice and the BSL1, BSL2, or BSL3transcript is detected in Northern blots using oligonucleotide probesspecific for the transcript. For protein expression detection,antibodies that are specific for the BSL1, BSL2, or BSL3 polypeptide areused, for example, in flow cytometric analysis, immunohistochemicalstaining, and activity assays. Alternatively, functional assays areperformed using preparations of different cell types collected from theknockout mice.

Several approaches can be used to produce transgenic mice. In oneapproach, a targeting vector is integrated into ES cell by homologousrecombination, an intrachromosomal recombination event is used toeliminate the selectable markers, and only the transgene is left behind(A. L. Joyner et al. (1989) Nature 338(6211):153-6; P. Hasty et al.(1991) Nature 350(6315):243-6; V. Valancius and O, Smithies, (1991) Mol.Cell. Biol. 11(3):1402-8; S. Fiering et al. (1993) Proc. Natl. Acad.Sci. USA 90(18):8469-73). In an alternative approach, two or morestrains are created; one strain contains the gene knocked-out byhomologous recombination, while one or more strains contain transgenes.The knockout strain is crossed with the transgenic strain to produce newline of animals in which the original wild-type allele has been replaced(although not at the same site) with a transgene. Notably, knockout andtransgenic animals can be produced by commercial facilities (e.g., TheLerner Research Institute, Cleveland, Ohio; B&K Universal, Inc.,Fremont, Calif.; DNX Transgenic Sciences, Cranbury, N.J.; IncyteGenomics, Inc., St. Louis, Mo.).

Transgenic animals (e.g., mice) containing a nucleic acid molecule whichencodes human BSL1, BSL2, or BSL3, may be used as in viva models tostudy the effects of altered expression of BSL1, BSL2, or BSL3. Suchanimals can also be used in drug evaluation and discovery efforts tofind compounds effective to inhibit or modulate the activity of BSL1,BSL2, or BSL3, such as for example compounds for treating immune systemdisorders, diseases, or conditions. One having ordinary skill in the artcan use standard techniques to produce transgenic animals which producehuman BSL1, BSL2, or BSL3 polypeptide, and use the animals in drugevaluation and discovery projects (see, e.g., U.S. Pat. No. 4,873,191 toWagner; U.S. Pat. No. 4,736,866 to Leder).

In another embodiment of the present invention, the transgenic animalcan comprise a recombinant expression vector in which the nucleotidesequence that encodes human BSL1, BSL2, or BSL3 is operably linked to atissue specific promoter whereby the coding sequence is only expressedin that specific tissue. For example, the tissue specific promoter canbe a mammary cell specific promoter and the recombinant protein soexpressed is recovered from the animal's milk.

In yet another embodiment of the present invention, a BSL1, BSL2, orBSL3 “knockout” can be produced by administering to the animalantibodies (e.g., neutralizing antibodies) that specifically recognizean endogenous BSL1, BSL2, or BSL3 polypeptide. The antibodies can act todisrupt function of the endogenous BSL1, BSL2, or BSL3 polypeptide, andthereby produce a null phenotype. In one specific example, a murineBSL1, BSL2, or BSL3 polypeptide or peptide can be used to generateantibodies. These antibodies can then be given to a mouse to knockoutthe function of the corresponding mouse protein.

In addition, non-mammalian organisms may be used to study BSL1, BSL2, orBSL3, and their related diseases. For example, model organisms such asC. elegans, D. melanogasfer, and S. cerevisiae may be used. BSL1, BSL2,or BSL3 homologues can be identified in these model organisms, andmutated or deleted to produce a BSL1-, BSL2-, or BSL3-deficient strain.Human BSL1, BSL2, or BSL3 can then be tested for the ability to“complement” the deficient strain. BSL1-, BSL2-, or BSL3-deficientstrains can also be used for drug screening. The study of BSL1, BSL2, orBSL3 homologs can facilitate the understanding of the biologicalfunction corresponding human gene, and assist in the identification ofbinding proteins (e.g., agonists and antagonists).

EXAMPLES

The examples as set forth herein are meant to exemplify the variousaspects of the present invention and are not intended to limit theinvention in any way.

Example 1 Identification of BSL1

Preparation of Monocytes:

Human monocytes were obtained from peripheral blood mononuclear cells byelutriation. The elutriation buffer contained 1 liter RPMI(GibcoBRL/Life Technologies Inc., Rockville, Md.; Cat. #11875-085) with2.5 mM EDTA and 10 μg/ml polymyxin B. The buffer was prepared 1 dayprior to the elutriation procedure, and stored at room temperature. Forthe elutriation procedure, 225 ml EDTA whole blood/donor was obtainedand prepared. Twenty milliliters of elutriation buffer was added totwelve 50 ml centrifuge tubes, and the 225 ml blood was divided equallyamong the tubes. Twelve milliliters of ficoll solution (lymphocyteseparation medium) was used to underlay the mixture in each tube(AccuPrep Lymphocytes, Accurate Scientific Co.; Cat. #1053980). Thetubes were centrifuged at 1800 rpm for 25 min (minutes).

Sheep's red blood cells were prepared by the following procedure. Twelvemilliliters of Sheep Blood Alsever's (SRBC; Colorado Serum Co., Denver,Colo.; Cat. #CS1112) was resuspended, and centrifuged at 2000 rpm for 10min. The top coating of the SRBC pellet was removed, and the pellet waswashed 2 times with elutriation buffer. Following each wash, the pelletwas centrifuged at 2200 rpm for 8 min. After the second wash, the pelletwas resuspended in 5 ml elutriation buffer and refrigerated.

Following centrifugation, the supernatant of the ficoll underlay tubeswas aspirated to leave behind approximately 5 ml of the interface layer.The interface layer was then carefully removed to a new tube, withoutdisturbing the red cell pellet in each tube. The interface layers of two50 ml tubes was combined and transferred to a new 50 ml tube, resultingin a total of six 50 ml tubes. Elutriation buffer was added to each tubeto bring final volume to 50 ml per tube. The tubes were centrifuged at1800 rpm for 10 min, and the supernatant was removed. The peripheralblood monocytes from each donor was combined and divided between twotubes, and resuspended in 40 ml elutriation buffer per tube. The tubeswere centrifuged at 1500 rpm for 10 min, the supernatant was aspirated,and the pellet was resuspended in 30 ml elutriation buffer per tube.

Following this step, 3 ml of washed SRBC was added to each tube, and themixture was centrifuged at 1000 rpm for 5 min. The pellet was incubatedon ice for at least 1 hr, and each pellet was gently resuspended byinverting the tubes. Twelve milliliters of ficoll was used to underlaythe mixture in each tube. The tubes were centrifuged at 2500 rpm for 20min, and the interface layers were removed to new tubes as previouslydescribed. The interface layers were then resuspended in 10 mlelutriation buffer and stored on ice until the start of the elutriationprocedure.

Elutriation of Monocytes:

Elutriation was performed as follows. The elutriation pump speed was setto ˜65 ml/min, and the storage ethanol was removed from the feed linesfor the pump. The feed lines, chambers, and rotor were washed with ˜100ml distilled water. Pump speed was reduced to 50 ml/min, and elutriationbuffer was passed through the feed lines, chamber, and rotor. The feedlines were checked for the presence of bubbles, and any bubbles wereremoved. The centrifuge speed was then set at 1950±1 rpm, and the pumpwas calibrated at 15 ml/min. The pump speed was then reduced to 11ml/min and the stopcock to the chamber was closed.

To load cells, the loading syringe stopcock was closed and the outletpipet was placed in the 50 ml sample tube labeled as 11 ml/min fraction.The cells were mixed and added to the loading syringe. The stopcock ofthe loading syringe was opened and the feeding tube was rinsed with 10ml elutriation buffer. When ˜0.5 ml cells remained in the syringe,elutriation buffer was added and this step was repeated one more time.Following this step, the loading syringe stopcock was closed and thechamber stopcock was opened.

Fifty milliliter fractions were collected at 11 (2 fractions), 14, 16,and 36 (2 fractions) ml/min pump speeds, and then placed on ice. Inaccordance with previous observations, the monocytes were predicted tobe in the 36 ml/min fraction. The monocyte fraction was centrifuged at1800 rpm for 8 min, and the cells were resuspended in 10 ml 25% FetalBovine Serum/RPMI with 1 μg/ml polymyxin B. The cells were counted andstored on ice until use.

Cell Culture Conditions:

The isolated peripheral blood monocytes were resuspended in RPMI 1640medium containing 10% fetal bovine serum (Hyclone, Logan, Utah)supplemented with penicillin/streptomycin, 2 mM glutamine, 1%non-essential amino acids, 1 mM sodium pyruvate, and IL-4 (75 ng/ml) andGM-CSF (15 ng/ml) cytokines (each from GibcoBRL). The cell suspension(5×10⁵ cells/ml) was transferred to tissue culture flasks and incubatedin chambers containing 5% CO₂ at 37° C. for 7 days. Following theincubation period, the cell cultures were pipetted vigorously to removecells from the flask. The human monocytic cell line THP1 was grown at 37C and 5% CO₂ to a final concentration of 5×10⁵ cells/ml in RPMI 1640medium containing 10% fetal bovine serum supplemented withpenicillin/streptomycin and 2 mM glutamine.

Poly(A)⁺ RNA Isolation:

GM-CSF/IL-4 differentiated human peripheral blood mononuclear cells(2×10⁸ cells) and THP1 human monocytes (2×10⁸ cells) were washed twicewith PBS (GibcoBRL) at 4° C. Poly(A)⁺ RNA was isolated directly usingthe FAST TRACK™ 2.0 kit (Invitrogen Corp., Carlsbad, Calif.).

Subtraction Library Construction:

A cDNA subtraction library was made using the CLONTECH PCR-Select™ cDNASubtraction Kit (CLONTECH, Palo Alto, Calif.). Manufacturer's protocolswere followed using 2.0 μg of GM-CSF/IL-4 differentiated humanperipheral blood monocyte poly (A)⁺ RNA as the tester sample and 2.0 μgof resting THP1 monocyte poly(A)⁺ RNA as the driver sample. Tensecondary PCRs were combined and run on a 1.2% agarose gel. Fragmentsranging from approximately 0.3 kb to 1.5 kb were gel purified using theQIAGEN gel extraction kit (QIAGEN, Valencia, Calif.) and inserted intothe TA CLONING® vector, pCR2.1 (Invitrogen). TOP10F′ competent E. coli(Invitrogen) were transfected and plated onto Lauria-Bertani (LB) platescontaining 50 μg/ml ampicillin. Approximately 300 clones were isolatedand grown in LB broth containing similar concentrations of ampicillin.Plasmids were isolated using QIAGEN miniprep spin (QIAGEN) and sequencedusing ABI cycle sequencers (ABI PRISM®, PE Applied Biosystems).

Full-Length Cloning:

To clone the 5′ and 3′ ends of BSL1, the SMART™ RACE (rapidamplification of cDNA ends) cDNA Amplification kit (CLONTECH) was usedaccording to the manufacturer's directions. The 5′ and 3′ RACE librarieswere constructed using 1.0 μg of poly(A)⁺ RNA template obtained fromhuman microvascular endothelial cell treated with TNF-alpha for 1 hr.The 5′-RACE-PCR mixture contained 1.5 μl of 5′-RACE ready cDNA, 0.4 μMJNF 155 primer (5′-GGCATAATAAGATGGCTCCC-3′) (SEQ ID NO:21), 1× UniversalPrimer Mix (UPM), 200 μM dNTP, 1× Advantaq Plus PCR buffer (CLONTECH),and 1× Advantaq Plus Polymerase (CLONTECH) in a total volume of 25 μl.The 3′-RACE-PCR mixture contained the same buffer conditions, 3′-RACEready cDNA, and 0.4 μM JNF 154 primer (5′-CATGAACTGACATGTCAGGC-3′) (SEQID NO:22). Both reactions were incubated using a traditional touchdownPCR approach: 5 cycles of incubation at 94° C. for 30 sec (seconds), 65°C. for 30 sec, and 72° C. for 3 min; 5 cycles of incubation at 94° C.for 30 sec, 63° C. for 30 sec, and 72° C. for 3 min; and 15 cycles ofincubation at 94° C. for 30 sec, 62° C. for 30 sec, and 72° C. for 3min.

The PCR products were isolated by electrophoresis using a 2.0% agarosegel, and DNA was visualized by ethidium bromide staining. An 888-bpfragment from the 5′-RACE reaction and a 1,110-bp fragment from the3′-RACE reaction were purified using the QIAGEN gel extraction kit andresuspended in 10 μl distilled water. Six microliters of each fragmentwas ligated into pCR2.1—TA CLONING® vector (Invitrogen), and theligation mixture was used to transfect TOP10F′ ultracompetent E. colicells (Invitrogen). Transfected cells were plated onto LB platessupplemented with 50 μg/ml ampicillin, 40 mg/ml X-gal, and 100 mM IPTG.Colonies were isolated and grown overnight at 37° C. in 4 ml of LB-brothsupplemented with 50 μg/ml ampicillin. Plasmids were isolated using theQIAGEN miniprep spin kit (QIAGEN), resuspended in 30 μl distilled water,and sequenced using an ABI cycle sequencer (ABI PRISM®, PE AppliedBiosystems).

To generate the full-length clone, JNF155RACE5.1, JNF154RACE3.2, andpCR2.1 were ligated together. JNF155RACE5.1 was doubly digested withXhoI and HindIII. JNF154RACE3.2 was doubly digested with HindIII andEcoRI. The TA CLONING® vector (Invitrogen) was digested with XhoI andEcoRI. Each fragment was purified using the QIAGEN gel extraction kitand resuspended in water. One microliter of each digested fragment wasligated together in the same reaction using T4 DNA ligase. The ligationmixture was used to transfect TOP10F′ E. coli ultracompetent cells,plated onto LB plates containing 50 μg/ml ampicillin, 40 mg/ml X-gal,and 100 mM IPTG. Colonies were isolated from the plates, and grown in LBbroth containing 50 μg/ml ampicillin overnight at 37° C. Plasmidscontaining full-length BSL1 were purified using the QIAGEN miniprep spinkit (QIAGEN), and sequenced (ABI cycle sequencer; PE AppliedBiosystems). The plasmid carrying DNA encoding the full-length BSL1sequence (pTADV:BSL1) was deposited with the American Type CultureCollection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209 USA),under ATCC Designation No. PTA-1989, on Jun. 6, 2000.

Example 2 Characterization of BSL1

Sequence Analysis of the BSL1 Clones:

The full-length BSL1 nucleotide and predicted amino acid sequence wasdetermined from a clone isolated from TNF-alpha treated humanmicrovascular endothelial cell cDNA subtraction library (FIGS. 1A and1B) and a clone isolated from a GM-CSF/IL-4 differentiated humanmonocyte cDNA library (FIG. 1C). The sequencing primers for the BSL1clones are shown in Table 1.

TABLE 1 SEQ ID Primer Sequence NO: JNF 292 forward CATTTACAAAGAGAGGTCGG23 JNF 298 reverse AGGGTTATTTTAAGTACCGACC 24 JNF 293 forwardGGAAATGTATGTTAAAAGCACG 25 JNF 297 reverse GGCATGGATCCTCAGCCCTGGG 26JNF 294 forward GAGACCCATGGGCTCTCCAGGG 27 JNF 296 reverseGTTCAAGCACAACGAATGAGGC 28 JNF 295 forward TGGCTTTGCCACATGTCAAGGC 29

Both BSL1 clones had identical coding sequences, however, the cloneobtained from the differentiated human monocyte cDNA library contained adifferent sequence in the 3′ untranslated region of the BSL1 gene (FIG.2B; see bold text). The nucleotide and predicted amino acid sequences ofBSL1 are shown in FIGS. 1A-1C.

EST clones encoding BSL1 were identified from public (GENBANK®) andprivate (Incyte Genomics) databases, and are shown in Table 2.

TABLE 2 Data- Length Position Clone ID base Tissue (bp) (1-3797)A1733919 GenBank Ovary tumor 429 401-829 AA292201 GenBank Ovary tumor430 401-830 AA399416 GenBank Ovary tumor 325 506-830 3166966H1 IncyteCD4⁺ T lymphos t/CD3, 197 415-611 CD28 Ab’s 4415633H1 Incyte PeripheralBlood 253 542-794 Monocytes, t/anti-IL-10, LPS AA368815 GenBankPlacenta, fetal 55  998-1052 5611256H1 Incyte Peripheral Blood 2541005-1258 Monocytes, t/anti-IL-10, LPS, SUB 5048659F6 Incyte Placenta,fetal 332 1016-1347 3680369H1 Incyte Lung, aw/asthma 240 1203-1442Al202916 GenBank Germ cell tumor, 259 1381-1639 pool, SUB AA373164GenBank Lung fibroblast line, 274 1416-1689 HSC172, fetal 4354914H1Incyte Fat, auxiliary, aw/ 288 1449-1736 breast adenoCA AA037078 GenBankFibroblasts, senescent 365 1529-1893 171033R6 Incyte Bone Marrow 2731785-2057 R30906/ GenBank Placenta, neonatal 1932 1867-3798 R30861**Full-length sequence not known.

It is noted that the BSL1 coding sequence and predicted amino acidsequence have also been identified as B7-H1 and PD-L1 (H. Dong et al.(1999) Nature Med. 5:1365-9; GenPept Accession No. NP_(—)054862; G. J.Freeman et al. (2000) J. Exp. Med. 192:1027-1034). In addition, a murinehomolog of B7-H1 has been identified (H. Tamura et al. (2001) Blood97:1809-1816). Notably, the mouse and human B7-H1 factors have beendescribed as costimulatory molecules (H. Dong et al. (1999) Nature Med.5:1365-9; H. Tamura et al. (2001) Blood 97:1809-1816), whereas PD-L1 hasbeen described as an inhibitor of T-cell proliferation (G. J. Freeman etal. (2000) J. Exp. Med. 192:1027-1034).

Chromosomal Mapping:

BSL1 was previously mapped utilizing radiation hybrid mapping (T. Ishidaet al. (1999) CytoGenet. Cell Genet. 85:232-6). Analysis of NCBI'sGenemap '99 using GENBANK® EST AA399416 indicated that the BSL1 gene waslinked to chromosome 9p24 with the order of AFM274xe1-stSG46389(BSL1)-AFM242xh6.

BSL1 Expression Analysis:

BSL1 expression patterns were determined by northern blot analysis ofseveral cell types, including resting peripheral blood T-cells,peripheral blood T-cells stimulated with anti-CD3/anti-CD28 antibodies,peripheral blood T-cells stimulated with phorbol 12 myristate 13 acetate(PMA), peripheral blood T-cells stimulated with phytohemaglutinin (PHA),resting THP1 monocytes, THP1 stimulated with lipopolysacharide (LPS),resting peripheral blood monocytes, resting peripheral blood monocytesstimulated with PHA, resting peripheral blood monocytes stimulated withGM-CSF and IL-4, RAJI B cells, RAMOS B cells, resting humanmicrovascular endothelial cells (HMVEC), HMVEC stimulated withTNF-alpha, and serum starved H292 human lung epithelial cells.

Cell Culture Conditions:

Peripheral blood T-cells were grown in RPMI 1640 (Hyclone) with 10%human serum at 37° C. and 5% CO₂ for 48 hr. Peripheral blood T-cellsstimulated with anti-CD3 and anti-CD28 antibodies were grown in RPMI1640 with 10% human serum at 37° C. and 5% CO₂ for 24-72 hr in thepresence of 1 μg/ml anti-CD3 monoclonal antibodies (P. S. Linsley et al.(1993) Ann. Rev. immunol. 11:191-212) and 1 μg/ml anti-CD28 monoclonalantibodies (Linsley et al., supra). Peripheral blood T-cells stimulatedwith PMA and ionomycin were grown in RPMI 1640 (Hyclone) with 10% humanserum at 37° C. and 5% CO₂ for 48 hr in the presence of 30 ng/ml PMAwith 1 μM ionomycin. Peripheral blood T-cells stimulated with PHA weregrown in RPMI 1640 (Hyclone) with 10% human serum at 37° C. and 5% CO₂for 48 hr in the presence of 3 μg/ml PHA. THP1 cells obtained from animmortal human monocytic cell line were grown in RPMI 1640 (Hyclone)with 10% fetal bovine serum at 37° C. and 5% CO₂ with or without 100ng/ml LPS for 2 hr. Peripheral blood monocytes were grown in RPMI 1640(Hyclone) with 25% fetal bovine serum in teflon plates at 37° C. and 5%CO₂ with or without 1 μg/ml PHA or 15 ng/ml GM-CSF with 75 ng/ml IL-4for 7 days. RAJI and RAMOS cells obtained from immortal human B celllines were grown in RPMI 1640 (Hyclone) with 10% fetal bovine serum at37° C. and 5% CO₂. HMVEC were grown in DMEM with 10% fetal bovine serumat 37° C. and 5% CO₂ with or without 10 ng/ml TNF-alpha for 1-24 hr.H292 cells obtained from an immortal human lung epithelial cell linewere grown in RPM1 1640 (Hyclone) with 10% fetal bovine serum at 37° C.and 5% CO₂, and then grown in serum free medium for 16 hr prior toharvest.

Northern Blot Analysis:

For northern blot analysis, 0.5 μg of total poly(A)⁺ RNA obtained fromeach cell type was separated on a 1.2% agarose gel containing 3%formaldehyde, and transferred to a HYBOND®—N+ nylon membrane (Amersham)overnight using 20×SSC as transfer buffer. The membrane was thenauto-crosslinked, washed with 4×SSPE, and allowed to air-dry. Themembrane was then prehybridized at 65° C. in ExpressHyb solution(CLONTECH) for 1 hr, and then hybridized with a [³²P]dCTP-radiolabeled(NEN, Boston, Mass.) random primed BSL1 cDNA probe. The probe wasobtained from a 666 bp BSL1 HindIII/PstI fragment (FIG. 6A), which waspurified using the NUCTRAP® purification column (Stratagene), andradiolabeled to have a specific activity of 2.0×10⁶ cpm/ml. Followinghybridization, the membrane was washed in 2.0×SSC with 0.05% SDS at 65°C., and exposed to film for 72 hr at −70° C.

A 3.8 kb BSL1 mRNA transcript was detected in several cell types. Inparticular, high levels of BSL1 mRNA were detected in peripheral bloodmonocytes stimulated with PHA, and in HMVEC stimulated with TNF-alpha(FIG. 7D). Moderate levels of BSL1 mRNA were detected in peripheralblood T-cells following stimulation with anti-CD3 and anti-CD28monoclonal antibodies for 72 hr (FIG. 7D). Moderate levels of BSL1 mRNAwere also observed in THP1 cells stimulated with LPS (FIG. 7D). However,BSL1 mRNA was not detected in resting THP1 cells, resting BJAB cells,LPS-activated BJAB cells, resting peripheral blood T-cells,PBT-activated peripheral blood T-cells, or GM-CSF/IL-4-activatedperipheral blood monocytes (FIG. 7D). In addition, BSL1 mRNA was notdetected in resting RAJI cells, resting RAMOS cells, or serum starvedH292 cells (FIG. 7D).

BSL1-Ig Fusion Construct:

The DNA fragment corresponding to the BSL1 predicted extracellulardomain (ECD; amino acids 23-290) was amplified by PCR utilizingfull-length BSL1-pCR2.1 as a template, and oligonucleotide primers thathybridize to the 5′ and 3′ ends of the BSL1 ECD: JNF 184 forward primer5′-TCAGGTACTAGTGTT CCCAAGGACCTATATGTGG-3′) (SEQ ID NO:30); and JNF 185reverse primer (5′-GATTCGAGATCTCCTCGAGTCCTTTCATTTGGAGGATGTGC C-3′ (SEQID NO:31). PCR was performed using ˜100 ng template DNA, 0.4 μM of eachprimer, 200 μM dNTP, 1× ADVANTAGE® 2 PCR buffer, and 1× ADVANTAGE® 2Polymerase (CLONTECH) in a total volume of 50 The PCR mixture wasincubated at 94° C. for 30 sec, 62° C. for 30 sec, and 72° C. for 1 min,and this was repeated for 30 cycles. The PCR products were separated bygel electrophoresis on a 1.2% agarose gel, and the DNA was visualized byethidium bromide staining. A 680-bp fragment corresponding to the BSL1ECD was purified from the agarose gel using the QIAGEN gel extractionkit (QIAGEN), and the resuspended in 32 μl distilled water.

The BSL1 ECD fragment was then digested using SpeI and BglII restrictionendonucleases and directionally cloned into SpeI/BamHI-digested PD19vector using 5 U/μl T4 DNA ligase (GibcoBRL). The ligation mixture wasused to transfect DH5 alpha competent E. coli cells (GibcoBRL), andtransfected cells were plated onto Lauria-Bertani (LB) plates containing50 μg/ml ampicillin. Plates were incubated overnight at 37° C., andcolonies were isolated and grown overnight at 37° C. in LB brothcontaining 50 μg/ml ampicillin. Plasmids were isolated using the QIAGENminiprep spin kit, resuspended in 50 μl distilled water, and sequencedusing ABI cycle sequencer (PE Biosystems, Foster City, Calif.). Primersequences were as follows: sense JNF 184 (5′-TCAGGTACTAGTGTTCCCAAGGACCATATGTGG-3′) (SEQ ID NO:32) and anti-sense JNF 185(5′-GATTCGAGATCTCCTCGAGTCTTTCATTGGGGATGTGCC-3′) (SEQ ID NO:33).

The plasmid carrying DNA encoding BSL1-Ig (pD19:BSL1Ig) was depositedwith the American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209 USA), under ATCC Designation No. PTA-1992, onJun. 6, 2000. The nucleotide and predicted amino acid sequence ofBSL1-Ig is shown in FIGS. 2A and 2B.

BSL1 Monoclonal Antibodies:

The BSL1-Ig fusion protein was purified by affinity purification asdescribed for BSL3-Ig, below. The purified fusion protein was then usedto immunize mice using the protocol described for BSL3-Ig. Followingthis, hybridoma cell lines were constructed and BSL1 monoclonalantibodies (MAbs) were isolated as described for BSL3, below. Inaddition, BSL1 MAbs were screened for specificity by whole cell ELISA.For whole cell ELISA, COS cells were transiently transfected with fulllength BSL1 (L156-3). Cells were lifted with VERSENE® on day 4 followingtransfection. Cells were washed twice in PBS and resuspended in PBS with10% FBS at a concentration of 5.0×10⁶ cells/ml. Then, 50 μl of cellswere added to each well of a Falcon 3911 96-well plate and incubated onice for 30 min. Next, 50 μl supernatant from the putative BSL1hybridomas was added per well and incubated on ice 30 min. Cells werewashed twice in PBS. Cells were then resuspended in goat anti-mouseHRP-conjugated secondary antibodies (Amersham Cat. #NA9310) diluted1:1000 in PBS. Cells were incubated on ice for 30 min, washed twice inPBS, and resuspended in 125 μl PBS. Following this, cells weretransferred to a fresh plate. Cells were washed in PBS and resuspendedin 25 μl PBS. Next, 125 μl Peroxidase solution B (KPL, Gaithersburg,Md.; Cat. #50-65-00) with TMB peroxidase substrate (KPL Cat. #50-76-01)was added. Color was allowed to develop. Cells were pelleted, and 100 μlsupernatant was transferred to an IMMULON® 2 plate. The signal wasquenched with 100 μl 1 N sulfuric acid, and the plates were read atOD₄₅₀/OD₆₃₀

Example 3 Identification of BSL2

Database Searches:

BSL2 was identified by BLAST and FASTA analysis of the Incyte Genomicssequence databases (Incyte Genomics) utilizing the B7-1 or B7-2 aminoacid sequences as query sequences. For BLAST analysis, the BLOSSUM-62scoring matrix was used (S. Henikoff et al. (1992) Proc. Natl. Acad.Sci, USA 89:10915-10919), and the remaining parameters were set to thedefault designations. For FASTA analysis, all the parameters were set tothe default designations (W. R. Pearson et al. (1988) Proc. Natl. Acad.Sci. USA 85:2444-2448). The sequence database searches identified twoIncyte Genomics ‘templates’: 252899.6 and the potential splice variant252899.8 (Incyte Genomics templates are consensus EST sequences that areconsidered to represent mRNA transcripts).

Sequence Analysis of the BSL2 Clone:

Incyte Genomics template 252899.8 was used to identify Incyte Genomicsclone 4616811. Incyte Genomics clone 4616811 belongs to Incyte GenomicsLibrary ID No. BRAYDIT01, which was originally constructed usingpoly(A)⁺ RNA from diseased hypothalamus tissue. Incyte Genomics clone4616811 was obtained from Incyte Genomics and used for sequence analysis(ABI cycle sequencer, PE Biosystems) with the primers shown in Table 3.

TABLE 3 SEQ ID Clone Primer Sequence NO: 4616811 392.423GGTGCACAGCTTTGCTGA 34 4616811 392.415 GCTGTGCACCAGCTGTTT 35 4616811392.439 GCTATGAAAGGTCCAGAG 36 4616811 392.499 GAATCTGGTGGTGTCCAA 374616811 392.1716 CTCTGTCACCATCACAGG 38 4616811 392.852CTCTGTCACCATCACACC 39 4616811 392.523 GAAATCCCGGATGCTCAC 40 4616811392.766A ACCACACGTGTTCCAGCA 41 4616811 392.766B TGCTGGAACACGTGTGGT 424616811 392.383 GGCCCTCAGCAAAGCTGT 43 4616811 392.1448AGCTGTAGGTGCCATTCG 44 4616811 392.892 AGGGACCTGGACCTCCAC 45 4616811392.1528 TGGGGGGAATGTCATAGG 46 4616811 392.1215 AGCAGGCAGGATGACTTA 474616811 392.1242 AACAGACCACCCACAACC 48 6487516 314.570GCAAATGGCACCTACAGC 49 6487516 314.634 TCTGGGGTGTGATGGTGA 50 6487516314.450 ATGAAAGGTCCAGAGGGC 51 6487516 314.584 ACCCATAATTCTTACCCA 526487516 314.824 CACAGCTCTGTTTGATCT 53 6487516 314.644 CTCCTACCCTCTGGCTGC54

Notably, the predicted amino acid sequence of Incyte Genomics clone4616811 contained 2 sets of V/C (variable/constant domain) folds,whereas typical B7-related amino acid sequences contain only 1 set ofV/C folds. SEQWEB® Gap (Genetics Computer Group) analysis indicated thatthe BSL2-4616811 sequence shared less than 50% sequence identity withB7-1, B7-2, BSL1/B7-H1 nucleotide sequences, while the BSL2-4616811amino acid sequence shared less than 35% sequence identity with theB7-1, B7-2, and BSL1/B7-H1 amino acid sequences. A sequence similar toBSL2-4616811 has been identified as an amyloid precursor protease inInternational Patent Application No. WO 00/68266 to G. W. Becker et al.

The nucleotide and predicted amino acid sequences of Incyte Genomicsclone 4616811 (BSL2-4616811) are shown in FIGS. 3A and 3B. The plasmidcarrying DNA encoding BSL2-4616811 (pINCY:BSL2-4616811) was depositedwith the American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209 USA), under ATCC Designation No. PTA-1993, onJun. 6, 2000.

Full-Length Cloning:

To verify the sequence of Incyte Genomics clone 4616811, PCR primerswere designed to amplify the nucleotide sequence from the predictedtranslation start codon to the predicted translation stop codon of theclone: forward primer BSL2-7 (5′-ATGCTGCGTCGGCG-3′) (SEQ ID NO:55);reverse primer BSL2-8 (5′-TCAGGCTATTTCTTGTCCATCATC-3′) (SEQ ID NO:56).

A HMVEC library was constructed utilizing the SMART™ RACE cDNAAmplification Kit (CLONTECH) according to manufacturer's instructions,using poly(A)⁺ RNA obtained from human microvascular endothelial cellstreated with TNF-alpha for 1 hr as the RACE reaction template. The PCRmixture included 1 μl PCR-ready HMVEC library, 5 μl PCR buffer(GibcoBRL), 1.5 μl 50 mM MgCl₂, 1 μl 10 mM dNTPs (Boehringer MannheimBiochemicals/Roche Molecular Biochemicals, Indianapolis, Ind.), 25 pMolBSL2-7 primer, 25 pMol BSL2-8 primer, and 1 μl CLONTECH ADVANTAGE®Enzyme mix in a total volume of 50 μl. PCR was performed in a PEBiosystems Thermal Cycler model 9700. The PCR mixture was incubated at94° C. for 1 min, followed by 35 cycles of incubation at 94° C. for 30sec, 60° C. for 30 sec, and 72° C. for 45 sec, followed by incubation at72° C. for 10 min.

One microliter of the PCR mixture was ligated directly into pCR2.1(Invitrogen) according to the manufacturer's directions. One half of theligation mixture was used to transfect MAX-EFFICIENCY® DH5 alpha E. colicells (GibcoBRL) in accordance with the manufacturer's directions.Transfected cells were plated onto LB agar plates with 100 μg/mlampicillin and 30 μg/ml X-gal and incubated at 37° C. overnight. Whitecolonies were isolated and grown overnight at 37° C. in 5 ml LB brothcontaining 100 μg/ml ampicillin.

Plasmid DNA was isolated from the bacterial culture using Spin Miniprepkit (QIAGEN) according to the manufacturer's directions. DNA wasdigested with EcoRI to release the cloned insert, and the digestionmixture was analyzed by electrophoresis on a 1% agarose gel. Insertfragments larger than 700 bp were sequenced using the vector-specificM13 (5′-GTTTTCCCAGTCACGAC-3′) (SEQ ID NO:57) and M13 reverse(5′-CAGGAAACAGCTATGAC-3′) (SEQ ID NO:58) sequencing primers (ABI cyclesequencer, PE Applied Biosystems).

Sequence analysis indicated that two splice variants of BSL2 had beencloned: BSL2-L165-21 and BSL2-L165-35b. The BSL2-L165-21 andBSL2-L165-35b splice variants encoded amino acid sequences that eachcontained one V/C fold and were ˜95% identical to one another. SEQWEB®Gap analysis (Genetics Computer Group) also indicated that theBSL2-L165-21 and BSL2-L165-35b nucleotide sequences shared less than 50%sequence identity with B7-1, B7-2, and BSL1/B7-H1 nucleotide sequences,while the BSL2-L165-21 and BSL2-L165-35b amino acid sequences sharedless than 35% sequence identity with the B7-1, B7-2, and BSL1/B7-H1amino acid sequences.

Sequence analysis further indicated that the amino acid and nucleotidesequences of BSL2-L165-21 shared less than 99% sequence identity withthe PRO352 amino acid and nucleotide sequences, respectively, reportedin International Patent Application No. WO 99/46281 to K. P. Baker etal. The amino acid and nucleotide sequences of BSL2-L165-35b shared lessthan 99.5% sequence identity with the PRO352 amino acid and nucleotidesequences, respectively.

Amino acid sequence alignments using GCG Gap program (GCG, Madison,Wis.) indicated that the longest stretch of identical amino acidresidues shared by BSL2-L165-21 and PRO352 was 88 contiguous amino acidsin length. The longest stretch of identical amino acid residues sharedby BSL2-L165-35b and PRO352 was 168 contiguous amino acids in length.The longest stretch of identical amino acids shared by BSL2-4616811 andPRO352 was 206 contiguous amino acids in length.

Nucleotide sequence alignments indicated that the longest stretch ofidentical bases shared by BSL2-L165-21 and PRO352 was 254 contiguousnucleotides in length. The longest stretch of identical bases shared byBSL2-L165-35b and PRO352 was 305 contiguous nucleotides in length. Thelongest stretch of identical bases shared by BSL2-4616811 and PRO352 was302 contiguous nucleotides in length. Notably, BSL2-L165-35b has alsobeen identified as B7-H3, a co-stimulatory molecule for T-cellactivation (A. I. Chapoval et al. (2001) Nature Immunology 2:269-274).

The nucleotide and predicted amino acid sequences of the BSL2-L165-21splice variant are shown in FIGS. 3C and 3D, while the nucleotide andpredicted amino acid sequences of BSL2-L165-35b are shown in FIGS. 3Eand 3F. The plasmid carrying DNA encoding BSL2-L165-21(pCR2.1:BSL2-L165-21) was deposited with the American Type CultureCollection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209 USA),under ATCC Designation No. PTA-1987, on Jun. 6, 2000. In addition, theplasmid carrying DNA encoding BSL2-L165-35b (pCR2.1:BSL2-L165-35b) wasdeposited with the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209 USA), under ATCC DesignationNo. PTA-1988, on Jun. 6, 2000.

Example 4 Characterization of BSL2

BSL2 Expression Analysis:

BSL2 expression patterns were determined by northern blot analysis ofvarious tissue and cell types, using the BSL2-4616811-derived probeshown in FIG. 6B, and the procedure described for BSL1 (see above). A3.6 kb BSL2 mRNA transcript was detected in several cell types. Inparticular, high levels of BSL2 mRNA were detected in all HMVECstimulated with TNF-alpha. Moderate levels of BSL2 mRNA were detected inresting THP1 cells, and THP1 cells activated with LPS (FIG. 7D). Incontrast, low levels of BSL2 mRNA were detected in peripheral bloodmonocytes stimulated with PHA or GM-CSF/IL-4, and BSL2 mRNA was notdetected in resting or stimulated peripheral blood T-cells, or inresting RAJI cells, resting RAMOS cells, or serum starved H292 cells(FIG. 7D).

PCR Assay to Determine Relative Abundance of BSL2-4616811 andBSL2-L165-35b:

To determine whether BSL2-4616811 or BSL2-L165-35b was predominantspecies of BSL2, and whether predominance corresponded with cell typeand/or stimulus type, the following experimental approach was used.Analysis of the genomic sequence of BSL2 indicated that the sequenceincludes several exons separated by introns. It was presumed that aprimary transcript was produced from this sequence, and the primarytranscript was spliced to yield BSL2-4616811 mature RNA. Analysis ofBSL2-4616811 sequence showed that it coded for the following: a 5′ UTR,an initiating ATG, a signal peptide sequence, a variable Ig fold (v1),an Ig constant fold (c1), an Ig V fold (v2), an Ig C fold (c2), a shorthinge a putative transmembrane domain, a short cytoplasmic tail, a stopcodon, and a 3′ UTR.

The BSL2-4616811 coding sequence appeared unique in the human genome, asthe v1 and c1 (v1c1) sequence was about 95% identical to the v2 and c2(v2c2) sequence at the amino acid level. Importantly, all of thestructurally important residues were conserved in the v1c1 and v2c2amino acid sequences, and most of the changes from v1c1 to v2c2 wereconservative changes. Comparison of the BSL2-L165-21 and BSL2-L165-35bsequences (which contained only single V and C folds) to theBSL2-4616811 sequence indicated that in each case, a splicing eventoccurred which resulted in a shift from v1 to the exactly homologousplace in v2. However, BSL2-L165-21 shifted from v1 to v2 at a differentplace than BSL2-L165-35b.

Despite the 96% identity between v1c1 and v2c2 at the nucleotide level,sequence comparison between the two regions using GCG Gap revealed oneshort region of relatively low homology. A forward primer designatedBSL2-9 was designed to take advantage of this short region of lowhomology. BSL2-9 was designed to bind specifically to v1 (FIG. 8Edemonstrates the specificity of the BSL2-9 primer). A reverse primer,BSL2-11 was designed to hybridize to the hinge sequence.

Notably, the BSL2-4616811 transcript contained both the BSL2-9v1-binding site in v1 and the homologous site in v2. The BSL2-L165-35btranscript contained only the BSL2-9 v1 binding site. The BSL2-L165-21transcript contained only the v2 site. Accordingly, PCR performed withprimers BSL2-9 and BSL2-11 was expected to produce: 1) a PCR product ofapproximately 1150 bp, representing BSL2-4616811; or 2) a PCR product ofabout 550 bp, representing BSL2-L165-35b.

PCR was initially conducted with BSL2-4616811 plasmid to confirm thespecificity of the BSL2-9 primer. PCR was performed using 2 μl 10 ng/μlBSL2-4616811 plasmid DNA, 5 μl PCR buffer (GibcoBRL), 1.5 μl of 25 mMMgCl₂ (GibcoBRL), 1 μl of 10 mM dNTPs (GibcoBRL), 2.5 μl of 10 pMol/μlBSL2-9 primer (5′ TGGTGCACAGCTTTGCT 3′) (SEQ ID NO:59), 2.5 μl of 10pMol/μl BSL2-11 primer (5′ TCTGGGGGGAATGTCAT 3′) (SEQ ID NO:60), 0.5 μlof 5 U/μl GibcoBRL PLATINUM® Taq DNA polymerase (Cat. #10966-018), and35 μl dH₂O. PCR was performed on a PE Biosystems 9700 using thefollowing cycling conditions: 94° C. for 30 sec; followed by 35 cyclesof 94° C. for 30 sec, 61° C. for 30 sec, 72° C. for 60 sec; followed by72° C. for 10 min. Following this, 40 μl of the PCR reaction was run ona 1.2% agarose gel next to Lambda BstEII DNA ladder (New England BiolabsBeverly, Mass.; Cat. #301-4S). The 1150 bp band was predominant,indicating that the BSL2-9 PCR primer was specific for the BSL2-4616811sequence (FIG. 8E).

Raji, Ramos, PM-LCL, PL-LCL, and CE-LCL B-like cell lines; HL-60 andThp1 monocytic like cell lines; and CEM and Hut78 T-like cell lines weregrown in RPMI 1640 with 10% FBS and 1% GibcoBRL penicillin/streptomycinto a concentration of 5×10⁵ cells/ml. The cultures were split andone-half of the B-like and T-like cell cultures were stimulated with 30ng/ml PMA and 1 μM ionomycin for 24 hr. Monocytic cell lines werestimulated with 1 μg/ml LPS for 24 hr. Early passage HUVEC were grown to90% confluence, and one-half of the culture was stimulated with 10 ng/mlTNFα, and harvested at 6 or 24 hr. Unstimulated HUVEC were harvested at24 hr.

Peripheral blood T-cells from two donors were purified as described,above. Cells were added (1×10⁶ cells/ml) to RPMI 1640 with 10% humanserum and 1% GibcoBRL penicillin/streptomycin. Cells were then incubatedat 37° C. and 5% CO₂ for 72 hr. T75 flasks were coated with 1 μg/ml CD3Mab G19.4 (described herein) in PBS for 4 hr at 37° C. The flask waswashed twice with PBS, and cells were added (1×10⁵ cells/ml) in RPMI1640 with 10% human serum and 1% GibcoBRL penicillin/streptomycin. CD28MAb 9.3 (previously described) was added to a final concentration of 1μg/ml. Cells were grown at 37° C. and 5% CO₂ for 72 hr. Cells were added(1×10⁶ cells/ml) to RPMI 1640 with 10% human serum and 1% GibcoBRLpenicillin/streptomycin. PMA was added to 30 ng/ml and ionomycin wasadded to 1 μM and incubated at 37° C. and 5% CO₂ for 48 hr.Proliferation of stimulated T-cells was confirmed visually. All celltypes were pelleted and frozen on dry ice and stored at −70° C. untiluse.

Total RNA was prepared using the Invitrogen (Carlsbad, Calif.) SNAPtotal RNA isolation kit (Cat. #K1950-01) according to the manufacturer'sinstructions. First strand cDNA was produced using the GibcoBRLSuperscript First-Strand Synthesis System for RT-PCR (Cat. #11904-018)according to the manufacturer's instructions for oligo dT priming. PCRwas performed using 2 μl first strand cDNA, 5 μl PCR buffer (GibcoBRL),1.5 μl of 25 mM MgCl₂ (GibcoBRL), 1 μl of 10 mM dNTPs (GibcoBRL), 2.5 μlof 10 pMol/μl BSL2-9 primer (5′ TGGTGCACAGCTTTGCT 3′) (SEQ ID NO:61),2.5 μl of 10 pMol/μl I BSL2-11 primer (5′ TCTGGGGGGAATGTCAT 3′) (SEQ IDNO:62), 0.5 μl of 5 U/μl GibcoBRL PLATINUM® Taq DNA polymerase (Cat.#10966-018), and 35 μl dH₂O. PCR was performed on a PE Biosystems 9700using the following cycling conditions: 94° C. for 30 sec; followed by35 cycles of 94° C. for 30 sec, 61° C. for 30 sec, 72° C. for 60 sec;followed by 72° C. for 10 min. Following this, 40 μl of the PCR reactionwas run on a 1.2% agarose gel.

PCR analysis indicated that certain cell types contained predominantlythe BSL2-4616811 transcript, with or without stimulation. Unstimulatedand stimulated PL-LCL cells showed higher levels of the BSL2-4616811transcript than the than the BSL2-L165-35b transcript (FIG. 8A). Bothunstimulated and stimulated HUVEC cells showed higher levels of theBSL2-4616811 transcript than the BSL2-L165-35b transcript (FIG. 8B).

In contrast, other cell types contained predominantly the BSL2-L165-35btranscript, with or without stimulation. Stimulated Raji cells, andunstimulated and stimulated Ramos cells showed higher levels of theBSL2-L165-35b transcript than the BSL2-4616811 transcript (FIG. 8A).Unstimulated and stimulated HL60 cells showed higher levels of theBSL2-L165-35b transcript than the BSL2-4616811 transcript (FIG. 8B).

In addition, certain cell types showed an increase in BSL2-4616811transcript levels upon activation. Unstimulated PM-LCL cells showedhigher levels of the BSL2-4616811 transcript, which increased relativeto the BSL2-L165-35b transcript upon stimulation (FIG. 8A). Similarly,unstimulated CE-LCL cells showed higher levels of the BSL2-4616811transcript, which increased relative to the BSL2-L165-35b transcriptupon stimulation (FIG. 8B). Unstimulated Thp1 cells showed equivalentlevels of the BSL2-4616811 and the BSL2-L165-35b transcript, however,levels of the BSL2-4616811 transcript increased upon stimulation (FIG.8B). Unstimulated peripheral blood T cells from donor 079 showedpredominantly BSL-L165-35b but shifted to predominantly BSL2-4616811upon stimulation. Peripheral blood T cells from donor 124 showed a lessdramatic shift from BSL2-L165-35b to BSL2-4616811 upon stimulation (FIG.8C). Unstimulated CEM cells showed higher levels of the BSL2-L165-35btranscript, but levels of the BSL2-4616811 transcript increased uponactivation (FIG. 8D).

Other cell types showed an increase in BSL2-L165-35b levels uponactivation. Unstimulated HUT78 cells showed higher levels of theBSL2-4616811 transcript, but levels of the BSL2-L165-35b transcriptincreased upon activation (FIG. 8D). These results, coupled with theconservation of the amino acid sequences in all four Ig folds, theconservation of structurally important amino acid residues in all fourIg folds, and the conservative nature of the amino acid differencesbetween v1c1 and v2c2, support a function for BSL2-4616811 (BSL2vcvc)which is distinct from BSL2-L165-35b and BSL2-L165-21 (BSL2vc).

To rule out the presence of PCR artifacts, the PCR product from HUVECactivated with TNFα for 24 hr was cloned using the Invitrogen OriginalTA CLONING® Kit (Cat. #K2000-01) according to the manufacturer'sdirections. The construct was used to transfect bacterial cells, and 25white colonies were isolated and grown in LB with 100 μg/ml ampicillin.DNA preps were made and sequencing was performed. Of the clones, 10clones did not contain insert, 3 clones did not produce readablesequence, 3 clones contained BSL2-related sequences with extensivedeletions, and the remaining 9 clones produced sequence consistent withBSL2-4616811.

It was noted that no PCR product corresponding to BSL2-4616811 orBSL2-L165-35b was observed in unstimulated Raji cells. To confirm thisresult, PCR was repeated for unstimulated and unstimulated Raji cells aspreviously described. Again, no PCR product was detected for theunstimulated Raji cells. Following this, PCR was performed using G3PDHprimers with template isolated from unstimulated and stimulated Rajicells. Cycling conditions were identical to those described above,except the annealing temperature was 55° C., and the extension time was45 sec. Relatively equal amounts of G3PDH product was detected for bothunstimulated and stimulated Raji cells. This provided further supportfor the observation that unstimulated Rail cells do not containdetectable BSL2 transcript.

BSL2-4616811-IQ Fusion Construct:

To construct the BSL2-4616811-Ig plasmid, the BSL2-4616811 extracellulardomain was PCR amplified from first strand cDNA (GibcoBRL Cat.#11904-018). cDNA was prepared from RNA purified from THP1 cellsstimulated with 100 ng/ml LPS for 2 hr. RNA was purified usingInvitrogen FASTTRACK® 2.0 (Cat. #K1593-02). The PCR reaction included 1μl cDNA; 5 μl GibcoBRL 10×PCR buffer; 1.5 μl 25 mM MgCl₂; 1 μl 10 mMdNTPs (Boehringer-Mannheim); 2.5 μl BSL2-L165-21-Ig-1 primer (10 pM/μl);2.5 μl BSL2-L165-21-Ig-2 primer (10 pM/μl); 1 μl CLONTECH ADVANTAGE®polymerase (Cat. #8417-1); and 35.5 μl milliQ H₂O. The primers containedthe following sequence: BSL2-L165-21-Ig-1: 5′ gg ggt acc ATG CTG CGT CGGCG 3′ (SEQ ID NO:63); BSL2-L165-21-Ig-2: 5′ cg gaa ttc TGG GGG GAA TGTCAT AG 3′ (SEQ ID NO:64). PCR samples were incubated at 94° C. for 1min, followed by 35 cycles of incubation at 94° C. for 30 sec, 59° C.for 30 sec, and 72° C. for 45 sec, followed by incubation at 72° C. for10 min.

Following this, 30 μl of the PCR reaction was run on a 1.2% agarose0.5×TBE gel. A band of approximately 1100 bp was excised and purifiedusing QIAGEN Gel Extraction Kit (Cat. #28704). One microliter of thepurified PCR product (L254) was ligated into pCR2.1 using the TACLONING® kit (Invitrogen; Cat. #K2050-01). Five microliters of theligation mixture was transfected into MAX-EFFICIENCY® DH5-alphacompetent bacteria (GibcoBRL; Cat. #18258-012), and transfected cellswere plated onto LB plates containing 100 μg/ml ampicillin and 800 μgX-Gal. White colonies were inoculated into 5 ml LB broth containing 100μg/ml ampicillin, and grown at 37° C. for 18 hr. Plasmid DNA waspurified with QIAGEN spin miniprep kit (Cat. #27106). Plasmid DNA wasdigested with KpnI and EcoRI. Plasmids containing inserts of about 1300bp were sequenced using Applied Biosystems automated DNA sequencers (ABI3700 capillary array sequencers). L254-7 was determined to containwild-type BSL2-4616811 sequence.

The Fc portion of human IgG1 was PCR amplified using 0.001 μl BSL1-Ig,described previously; 5 μl GibcoBRL PCR buffer; 1.5 μl 25 mM MgCl₂; 1 μl10 mM dNTPs (Boehringer-Mannheim); 2.5 μl Ig-1 primer (10 pM/μl) 2.5 μlBSL1Ig-2 primer (10 pM/μl); 1 μl CLONTECH ADVANTAGE® polymerase; 36 μldH₂O. The primers contained the following sequence: IgG-1: 5′ g gaa ttcGAG CCC AAA TCT TGT GAC AA 3′ (SEQ ID NO:65); BSL1Ig-2 gc gc tct aga TCATTT ACC CGG AGA CAG G (SEQ ID NO:66). PCR samples were incubated at 94°C. for 1 min; followed by 25 cycles of incubation at 94° C. for 30 sec,59° C. for 30 sec, and 72° C. for 30 sec; followed by incubation at 72°C. for 10 min.

Following this, 30 μl of the PCR reaction was run on a 1.2% agarose0.5×TBE gel. A band of about 700 bp was excised and purified usingQIAGEN QIAQUICK® gel extraction kit. Two microliters of the purifiedfragment (L174) was ligated into pCR2.1 using the TA CLONING® kit(Invitrogen). Five microliters of the ligation mixture was transfectedinto GibcoBRL MAX-EFFICIENCY® DH5-alpha competent bacteria, andtransfected cells were plated onto LB plates containing 100 μg/mlampicillin and 800 μg X-gal. Plates were incubated for 18 hr at 37° C.White colonies were inoculated into 5 ml LB broth containing 100 μg/mlampicillin and grown at 37° C. for 18 hr. Plasmid DNA was prepared usingQIAGEN QIAPREP® spin miniprep kit. Plasmid DNA was digested with EcoRIand run on an agarose gel. Plasmids containing inserts of about 900 bpwere sequenced. L174-3 was determined to contain wild-type human IgG1 Fcsequence.

L174-3 was digested with EcoRI/XbaI and separated on a 1.2% agarose0.5×TBE gel. A band of about 750 bp was excised and purified usingQIAGEN gel extraction kit. Ten microliters of the purified fragment wasrun on an agarose gel next to a standard to obtain an estimate of theconcentration. Approximately 20 ng of the EcoRI/XbaI fragment wasligated (Ligation 200) into 40 ng of EcoRI/XbaI digested pcDNA3.1+vector (Invitrogen) using GibcoBRL high concentration T4 DNA ligase (5U/μl) diluted in GibcoBRL T4 DNA ligase buffer. Five microliters of theligation mixture was transfected into MAX-EFFICIENCY® DH5-alphacompetent bacteria (GibcoBRL), and transfected cells were plated onto LBplates containing 100 μg/ml ampicillin. Plates were incubated at 37° C.for 18 hr. Colonies were inoculated into LB broth containing 100ampicillin. Plasmid DNA was purified using QIAGEN spin miniprep kit andsequenced. The L200-1 sequence was determined to be identical to theL174-3 sequence.

The L254-7 BSL2-4146811 construct was digested with KpnI/EcoRI. A bandof about 1300 bp was excised from a 1.2% agarose 0.5×TBE gel and ligatedinto the L200-1 Fc construct digested with KpnI/EcoRI. Five microlitersof the ligation reaction was transfected into MAX-EFFICIENCY® DH5-alphacells and plated onto LB plates containing 100 μg/ml ampicillin.Colonies were grown, and plasmid DNA was purified as above. Plasmid DNAwas digested with KpnI/XbaI and separated on an agarose gel as above.Plasmids containing a band of about 2 kb were sequenced as above.BSL2-4616811-Ig was determined to contain the wild-type BSL2-4616811 andwild-type human IgG1 sequences. The nucleotide and predicted amino acidsequence of BSL2-4616811-Ig is shown in FIGS. 4A and 4B.

BSL2 Monoclonal Antibodies:

The BSL2-4616811-Ig fusion protein was purified by affinity purificationas described for BSL3-Ig, below. The purified fusion protein was thenused to immunize mice using the protocol described for BSL3-Ig, exceptthat a fourth boost was used. Following this, hybridoma cell lines wereconstructed and BSL2 MAbs were isolated as described for BSL3, below.

Example 5 Identification of BSL3

Database Searches:

BSL3 was identified by BLAST and FASTA analysis of the Incyte Genomicssequence databases (Incyte Genomics) utilizing the B7-1 or B7-2 proteinsas query sequences, and the parameters described for the BSL2 searches.The sequence database searches identified Incyte Genomics ‘gene’ 117327(an Incyte Genomics gene is an EST sequence that is grouped with similarsequences, and considered to represent the product of a single genomiclocus). The Incyte Genomics gene 17327 has since been renamed as IncyteGenomics gene 899898. In a secondary screen, the BLAST and FASTAprograms were used to search the Incyte Genomics sequence databases(Incyte Genomics) for sequences related to the mouse AF142780 gene(potential ortholog of the 117327 gene), using the previously describedsearch parameters. These searches identified Incyte Genomics gene143522.

The 143522 and 117327 genes were then used to identify Incyte Genomicsclones 3844031 and 3207811, respectively. The 3844031 clone belongs toIncyte Genomics Library ID No. DENDTNT01, which was originallyconstructed utilizing poly(A)⁺ RNA isolated from untreated dendriticcells from peripheral blood. The 3207811 clone belongs to IncyteGenomics Library ID No. PENCNOT03, which was originally constructedutilizing poly(A)⁺ RNA isolated from corpus cavernosum tissue. The3844031 and 3207811 clones were obtained from Incyte Genomics, andsequenced (ABI cycle sequencer, PE Biosystems) using the primers shownin Table 4.

TABLE 4 SEQ ID Clone Primer Sequence NO: 3844031 316.491GAAGGCCTCTACCAGGTC 67 3844031 316.512 CTTTAGGCGCAGAACACT 68 3844031316.203 AAGGGTCAGCTAATGCTC 69 3844031 316.379 TCAGTTTGCACATCTGTA 703844031 316.1538 TATGCTATCAAGATTCCA 71 3844031 316.1839GTAAAGTGCAGTAGTGCT 72 3844031 316.1601 TATGAGCTCACAGACAGG 73 3207811315.560 AGGTTCAGATAGCACTGT 74 3207811 315.468 ACTTATCTGAAATTGCTG 753207811 315.493 TTGATATGCTCATACGTT 76 3207811 315.1011GAATTCTGTGGGTCCAGG 77 3207811 315.601 CATGTTTAATGGTGGTTT 78 3207811315.498 AAAGCTGTATTCTTCAAA 79

Full-Length Cloning of BSL3:

To clone the 5′ end of BSL3, the SMART™ RACE (rapid amplification ofcDNA ends) cDNA Amplification kit (CLONTECH) was used according to themanufacturer's directions. The 5′ RACE library was constructed using 1.0μg of poly(A)⁺ RNA template obtained from human microvascularendothelial cell treated with TNF-alpha for 1 hr. The 5′ RACE reactionmixture contained 2 μl RACE-ready cDNA, 1×PCR buffer (GibcoBRL), 200 μMdNTP (Boehringer Mannheim), 1.5 mM MgCl₂, 1 μl CLONTECH ADVANTAGE®enzyme mix, 1× CLONTECH SMART™ primer, and 25 pMol BSL3 3′ specificprimer (BSL3-2 5′-GAACACTGGTGACCTGGTAGAG-3′) (SEQ ID NO:80) in a totalvolume of 50 μl. The 5′ RACE reaction was performed in a GENEAMP® PCRSystem 9700 machine (PE Applied Biosystems) using an initialdenaturation step of incubation at 94° C. for 1 min, followed by 35cycles of incubation at 94° C. for 30 sec, 62° C. for 30 sec, 72° C. for30 sec, followed by incubation at 72° C. for 10 min.

The PCR products were analyzed by gel electrophoresis using a 1.2%agarose gel (GibcoBRL) with 0.5×TBE and 10 μg/ml ethidium bromide(Bio-Rad). An ˜875 bp fragment was excised from the gel and purifiedusing the QIAGEN QIAQUICK® Gel Extraction kit according to themanufacturers directions. One microliter of the purified fragment wasmixed with 2 μl pCR2.1 pTADV cloning vector (CLONTECH), 2 μl ligationcocktail (GibcoBRL), and 4 U T4 DNA ligase (GibcoBRL) in a total volumeof 10 μl, and the ligation mixture was incubated at 14 C for 4 hr. Fivemicroliters of the ligation mixture was used to transfectMAX-EFFICIENCY® DH5 alpha E. coli cells (GibcoBRL) according to themanufacturers directions, and transfected cells were plated onto LBplates containing 100 μg/ml ampicillin and 30 μg/ml X-gal. Whitecolonies were picked and grown in 5 ml of LB broth containing 100 μg/mlof ampicillin. DNA was purified from the bacteria using the QIAPREP®Spin Miniprep Kit (QIAGEN). Plasmid DNA was digested with EcoRI andanalyzed by agarose gel electrophoresis. Plasmid isolates containing anEcoRI fragment of approximately 900 bp were retained for sequencing (ABIcycle sequencer, PE Biosystems). The sequence was analyzed by SEQWEB®Gap (Genetics Computer Group).

The 5′ RACE library was then used as a template for PCR amplification.The PCR mixture contained 1 μg 5′ RACE library template, 1×PCR buffer(GibcoBRL), 200 μM dNTP (Boehringer Mannheim), 1.5 mM MgCl₂, 1 μlCLONTECH ADVANTAGE® enzyme mix, 25 pMol forward primer (BSL3-3:5′-CCGGGGTACCATGATCTTCCTCCTGCTAATGTTG-3′) (SEQ ID NO:81), and 25 pMolreverse primer (BSL3-4: 5′-GCGCTCTAGATCAGATAGCACTGTTCACTTCCC-3′) (SEQ IDNO:82) in a total volume of 50 μl. PCR was performed in a GENEAMP® PCRSystem 9700 (PE Applied Biosystems) machine using an initialdenaturation step of incubation at 94° C. for 1 min, followed by 30cycles of incubation at 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for30 sec, and followed by incubation at 72° C. for 10 min.

An ˜800 bp BSL3 PCR product was obtained. To clone the BSL3 fragment, 1μl of the PCR product was mixed with 2 μl pCR2.1 cloning vector(Invitrogen), 2 μl ligation buffer (GibcoBRL), 4 U T4 DNA ligase(GibcoBRL), and 4 μl H₂O. The ligation mixture was incubated at 14° C.for 4 hr, and 5 μl microliters of the mixture was used to transfectMAX-EFFICIENCY® DH5 alpha E. coli cells (GibcoBRL). Transfected cellswere plated onto LB agar plates containing 100 μg/ml ampicillin and 30μg/ml X-gal, and 18 white colonies were isolated and grown in 5 ml of LBbroth containing 100 μg/ml of ampicillin. DNA was purified from thebacterial culture using the QIAPREP® Spin Miniprep Kit (QIAGEN)according to the manufacturer's directions. Plasmid DNA was digestedwith EcoRI and analyzed by agarose gel electrophoresis. Sixteen plasmidisolates contained EcoRI fragment of ˜800 bp, and were retained forsequencing (ABI cycle sequencer, PE Biosystems) using thevector-specific M13 and M13 reverse primers (see above).

SEQWEB® Gap (Genetics Computer Group) analysis indicated the optimalalignment of the sequences of the BSL3 Incyte Genomics clones and theBSL3 5′ RACE product. SEQWEB® Gap analysis (Genetics Computer Group)also indicated that the BSL3 nucleotide sequence shared less than 55%sequence identity with B7-1, B7-2, or BSL1/B7-H1 nucleotide sequences,while the BSL3 amino acid sequence shared less than 45% sequenceidentity with the B7-1, B7-2, and BSL1/B7-H1 amino acid sequences.

In addition, the BSL3 C-terminal amino acid sequence shared less than98% identity to the amino acid sequence encoded by GENBANK® AccessionNo. AK001872, over a stretch of 174 amino acids. Amino acid sequencealignments using the GCG Gap program indicated that the longest stretchof identical residues shared by BSL3 and AK001872 was 99 contiguousamino acids in length. Nucleotide sequence alignments using GCG Gapindicated that the longest stretch of identical bases shared by BSL3 andAK001872 was 239 contiguous nucleotides in length. Notably, a mouseortholog of BSL3 was identified from the GENBANK® Database (AccessionNo. AF142780). The BSL3 N-terminal amino acid sequence sharedapproximately 70% sequence identity with the amino acid sequencecorresponding mouse AF142780, over a stretch of 250 amino acids. Inaddition, it was noted that BSL3 has also been identified as PD-L2, anapparent inhibitor of T-cell proliferation (Y. Latchman et al. (2001)Nature Immunology 2:261-268).

The nucleotide and predicted amino acid sequences of BSL3 are shown inFIGS. 5A and 5B. The plasmid carrying DNA encoding BSL3 (pCR2.1:BSL3)was deposited with the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209 USA), under ATCC DesignationNo. PTA-1986, on Jun. 6, 2000.

The BSL1, BSL2, and BSL3 sequence information is summarized in Table 5.

TABLE 5 Nucleic Amino Acid (NA) NA Acid (AA) AA BSL Sequence SEQ ID FIG.SEQ ID FIG. NO. Name NO: NO. NO: NO. 1 BSL1 (TNF-α) 1 1A 2 1B 1 BSL1(GM-CSF/IL-4) 3 1C 2 1B 1 BSL1-Ig 4 2A 5 2B 2 BSL2-4616811 6 3A 7 3B 2BSL2-L165-21 10 3C 11 3D 2 BSL2-L165-35b 12 3E 13 3F 2 BSL2-4616811-Ig 84A 9 4B 3 BSL3-L143 14 5A 15 5B 3 BSL3-L232-6-Ig 16 6A 17 6B

Example 6 Characterization of BSL3

BSL3 Expression Analysis:

BSL3 expression patterns were determined by northern blot analysis ofvarious cell types, using the BSL3 probe shown in FIG. 7C, and theprocedure described for BSL1. A 2.7 kb BSL3 mRNA transcript was detectedin several cell types. In particular, high levels of BSL3 mRNA weredetected in all HMVEC stimulated with TNF-alpha (FIG. 7D). Moderatelevels of BSL3 mRNA were detected in peripheral blood monocytesstimulated with PHA or GM-CSF/IL-4 (FIG. 7D). However, or BSL3 mRNA wasnot detected in any of the remaining cell types (FIG. 7D).

In addition, multiple tissue northern blots and expression arrays werepurchased from CLONTECH Laboratories and hybridized with P³²-labeledBSL3 probe. Briefly, a 900 bp BSL3 fragment (BSL3/KpnI+XbaI) wasisolated from clone L168-2 using KpnI and XbaI restrictionendonucleases, run on a 1.2% agarose gel, and purified using the QIAGENGel Extraction Kit. Approximately 30 ng of BSL3/KpnI+XbaI wasradiolabeled (6000 Ci/mmol P³²-dCTP) using the Random Primed DNALabeling Kit (Roche, Indianapolis, Ind.). Unincorporated nucleotideswere removed using NUCTRAP® Probe Purification Columns (Stratgene, LaJolla, Calif.). Radiolabeled BSL3/KpnI+XbaI probe was added at aspecific activity of 3.0×10⁶ cpm/ml of ExpressHyb hybridization solution(CLONTECH) and incubated overnight at 65° C. Blots were washed with0.1×SSC/0.1% SDS at 62° C. and exposed to film for 72 hr.

Northern blot analysis indicated that high levels of BSL3 transcriptwere present in spleen tissue; moderate levels of BSL3 transcript werepresent in thymus, testis, ovary, and small intestine tissues; and lowlevels of BSL3 transcript were present in heart, placenta, lung, liver,skeletal muscle, prostate, colon, lymph node, trachea, and adrenal glandtissues, and Burkitt's lymphoma Raji cell line (FIG. 7E). Microarrayanalysis indicated that high levels of BSL3 transcript were present inspleen tissue; moderate levels of BSL3 transcript were present in lung,liver, placenta, fetal spleen, lymph node, and fetal thymus tissues; andlow levels of BSL3 transcript were present in heart, aorta, corpuscallosum, left atrium, right atrium, jejunum, thymus, fetal liver, andmammary gland tissues (FIG. 7F).

Quantitative PCR:

BSL1 and BSL3 expression patterns were determined by quantitative PCR.Human Multiple Tissue cDNA (MTC™) Panels (Cat. #PT3158-1) were purchasedfrom CLONTECH. For each PCR reaction, 5 μl of cDNA was used. Human andmurine BSL1 and BSL3 PCR primers were designed by Primer3 program(Whitehead Institute for Biomedical Research; Steve Rozen, Helen J.Skaletsky, 1998, Primer3) as shown in Table 6.

TABLE 6 SEQ ID nucleo- Primer Sequence NO: tides human BSL1TACAAGCGAATTACTGTGAA 83 459-479 forward human BSL1 GATGTGCCAGAGGTAGTTCT84 773-793 reverse human BSL3 AATAGAGCATGGCAGCAATG 85 419-439; forwardhuman BSL3 GGCGACCCCATAGATGATTA 86 634-654 reverse human 18s rRNACCAGTAAGTGCGGGTCAT 87   7-25 forward human 18s rRNA TTCACCTACGGAAACCTT88 196-214 reverse

SYBR® Green PCR Core Reagents (Cat. #4306736) were purchased from PEApplied Biosystem. Real-time PCR was performed on ABI PRISM® 5700Sequence Detection System PE Applied Biosystem. PCR samples wereincubated at 95° C. for 15 sec, 55° C. for 20 sec, and 75° C. for 1 minfor 40 cycles. The BSL1 PCR product was 334 bp; the BSL3 PCR product was235 bp; the 18S rRNA PCR was 207 bp. Following PCR and data collection,dissociation curve studies were performed. In addition, PCR samples wereanalyzed by agarose gel electrophoresis to confirm the size of the PCRproduct.

Data Processing and Presentation:

For each PCR reaction, a threshold cycle number (C_(T)) was generated asa read-out by the real-time PCR machine. The data was processedaccording to the manual of ABI PRISM® 5700 Sequence Detection System.Briefly, the C_(T) of BSL1 and BSL3 was normalized to the C_(T) of 18SrRNA in the each sample. The data was then subtracted by the normalizedC_(T) in the sample showing lowest expression levels. For BSL1, thelowest expression levels were found in tonsil tissue. For BSL3, thelowest expression levels were found in skeletal muscle. The final datawas presented as the fold increase over the lowest expression levels.

Quantitative PCR indicated that high levels of BSL1 transcript werepresent in lymph node, spleen, lung, and placenta tissue; moderatelevels of BSL1 transcript were present in thymus and pancreas tissue;and low levels of BSL1 transcript were present in heart, brain, kidney,adult liver, skeletal muscle, bone marrow, leukocyte, fetal liver, andtonsil tissue (FIG. 7G). For BSL3, high levels of transcript werepresent in lymph node and spleen tissue; and low levels of BSL3transcript were present in thymus, lung, tonsil, heart, pancreas,placenta, kidney, bone marrow, fetal liver, and adult liver tissue(FIG.7H).

BSL3-Ig (L232-6) Fusion Construct:

The BSL3-Ig (L232-6) construct was made using the following procedure.The ECD of BSL3 was amplified by PCR using 0.01 μl pCR2.1:BSL3 (L143-4),described above; 5 μl GibcoBRL PCR buffer; 1.5 μl 25 mM MgCl₂; 1 μl 10mM dNTPs; 2.5 μl BSL3-3 primer (10 pM/μl); 2.5 μl BSL3Ig-6 primer (10pM/μl); 1 μl CLONTECH ADVANTAGE® polymerase; 36 μl dH₂O. The primerscontained the following sequence: BSL3-3: 5′ cc gg ggt acc ATG ATC TTCCTC CTG CTA ATG TTG 3′ (SEQ ID NO:89); BSL3Ig-6: 5′ cg gaa ttc GGT CCTGGG TTC CAT CTG 3′ (SEQ ID NO:90). PCR samples were incubated at 94° C.for 1 min; followed by 20 cycles of incubation at 94° C. for 30 sec, 59°C. for 30 sec, and 72° C. for 30 sec; followed by incubation at 72° C.for 10 min.

Following this, 38 μl of the PCR product was digested with KpnI/EcoRIand run on a 1.2% agarose 0.5×TBE gel. A band of about 650 bp wasexcised and purified with QIAGEN gel extraction kit. Ten microliters ofthe purified fragment was run on an agarose gel next to a size standard.Ten nanograms of fragment was ligated (L232) to 20 ng of KpnI/EcoRIdigested L200-1 (described previously). Five microliters of the ligationmixture was transfected into GibcoBRL MAX-EFFICIENCY® DH5 alphacompetent bacteria, and transfected cells were plated onto LB platescontaining 100 μg/ml ampicillin. Plates were incubated at 37° C. for 18hr. Colonies were inoculated into 5 ml of LB broth containing 100 μg/mlampicillin and grown for 18 hr at 37° C. DNA was purified using QIAGENspin miniprep kit and digested with PmeI. The digested samples were runon an agarose gel and plasmids that contained fragments of about 1500 bpwere sequenced. L232-6 was determined to have wild-type BSL3 sequence.The nucleotide and predicted amino acid sequence of BSL3-Ig (L232-6) isshown in FIGS. 6A and 6B.

BSL3-Iq (L275-1) Fusion Construct:

The BSL3-Ig (L275-1) construct was made using the following procedure.BSL3-Ig was PCR amplified from L232-6 using 0.001 μl L232-6; 5 μlGibcoBRL PCR buffer; 1.5 μl 25 mM MgCl₂; 1 μl 10 mM Boehringer-MannheimdNTPs; 2.5 μl BSL3-5 primer (10 pM/μl); 2.5 μl BSL1Ig-2 primer (10pM/μl); 1 μl CLONTECH ADVANTAGE® polymerase; and 35.5 μl dH₂O. Theprimers contained the following sequence: BSL3-5: 5′ cg gga ttc ATG ATCTTC CTC CTG CTA ATG TT 3′ (SEQ ID NO:91); BSL1Ig-2: 5′ gc gc tct aga TCATTT ACC CGG AGA CAG G 3′ (SEQ ID NO:92). PCR samples were incubated at94° C. for 1 min; followed by 20 cycles of incubation at 94° C. for 30sec, 58° C. for 30 sec, and 72° C. for 1.5 min; followed by incubationat 72° C. for 10 min.

Following this, 2 μl of the PCR reaction was ligated (L262) into pCR2.1using the TA CLONING® kit (Invitrogen). Five microliters of the ligationwas transfected into MAX-EFFICIENCY® DH5 alpha competent cells(GibcoBRL), and transfected cells were plated onto LB plates containing100 μg/ml ampicillin and 800 μg of X-Gal. Plates were incubated at 37°C. for 18 hr. White colonies were inoculated into 5 ml of LB brothcontaining 100 μg/ml ampicillin and grown at 37° C. for 18 hr. PlasmidDNA was purified using QIAGEN spin miniprep kit. Plasmid DNA wasdigested with BamHI/XbaI and analyzed on an agarose gel. Plasmids thatcontained an approximately 1300 bp fragment were sequenced. L262-2 wasdetermined to contain the wild-type BSL3 sequence.

L262-2 was digested with BamHI/XbaI and run on a 1.2% agarose 0.5×TBEgel. An approximately 1300 bp fragment was purified using QIAGEN gelextraction kit. Ten nanograms of the purified fragment was ligated into30 ng of BamHI/XbaI digested pD18 (related to pD16 and pD17 plasmids,described in U.S. Pat. No. 6,051,228). Five microliters of the ligationwas transfected into GibcoBRL MAX-EFFICIENCY® DH5 alpha competentbacteria. Transfected cells were plated onto LB plates containing 100μg/ml ampicillin and grown at 37° C. for 18 hr. Colonies were inoculatedinto 5 ml of LB broth containing 100 μg/ml ampicillin and grown at 37°C. for 18 hr. Plasmid DNA was purified using QIAGEN spin miniprep kit.Plasmid DNA was digested with BamHI plus XbaI or HindIII, and thedigested samples were analyzed on an agarose gel. As determined byrestriction mapping, L275-1 through L275-9 contained the correctconstruction.

Purification of BSL3-Ig Fusion Protein:

Purification of BSL3-Ig (human-IgG1) was accomplished by one-stepaffinity purification. Supernatant from transiently transfected COScells expressing BSL3-Ig was applied to a Sepharose column ofimmobilized protein A. The column was washed with PBS until theabsorbance at 280 nm reached the baseline level. Bound protein waseluted with Immuno Pure IgG Elution Buffer (Pierce Chemical, Rockford,Ill.; Cat. #21004). Fractions containing the bound protein wereneutralized with 1/8 v/v of 3 M sodium phosphate, pH 7. The resultingpreparation was dialyzed against PBS, filtered (0.2 μm). All bufferscontained 0.02% w/v sodium azide.

Immunization with BSL3 Polypeptide:

For the initial immunization, mice between 1 and 3 months were used(BalbC; Harlan, Indianapolis, Ind.). RIBI adjuvant was prepared asfollows. In one vial, 0.5 mg MPL (monophosphoryl lipid A; RIBIImmunochemical Research, Inc., Hamilton, Mont.); 0.5 mg TDM (synthetictrehalose dicorynomycolate; RIBI Immunochemical Research, Inc.); and 40μl Squalene with 0.2% TWEEN® 80 were mixed together. The mixture waswarmed to 40-45° C. for 5-10 min, and 2 ml of BSL3 polypeptide/PBS (125μg/ml) was added. The solution was vortexed vigorously for severalminutes. The solution was drawn into a syringe and injected immediatelyinto the mice.

Dosages followed recommendations by RIBI Immunochemical Research, Inc.For the first injection, approximately 100 μg of BSL3 polypeptide wasresuspended in 250 μl of 1×RIBI in PBS. The mixture was injectedintraperitoneally with 21 gauge needle. For second and later injections,boosts were given at least 3 weeks apart. Injections were at half dose.At least 3 weeks following the third injection, once the titer reachedan acceptable level (see below), the animal was given a final boost. Forfinal injections, approximately 1 mg/ml of BSL3 polypeptide wasresuspended in PBS (RIBI was omitted), and the mixture was administeredintravenously via tail veins. Animals were harvested 3-4 days later.

To monitor titer levels, sera samples were taken before initialimmunization (background) and 7-10 days after each immunization fortiter monitoring. Titer levels were measured by ELISA. Sera washarvested by eye-bleed, or tail-bleed. Typically, 200 μl of blood wasremoved for sera testing.

Hybridoma Cell Lines:

Hybridoma cell lines were constructed using the following reagents:Iscoves Modified Dulbecco's Medium (GibcoBRL; Cat. #12440-053); FetalBovine Serum (Hyclone; Cat. #A-1115L, Lot #11152564) heat inactivatedfor 30 min at 56° C.; L-Glutamine-200 mm, 100× (GibcoBRL; Cat.#25030-081); Penicillin/Streptomycin (GibcoBRL; Cat. #15140-122); ORIGENHybridoma Cloning Factor (Igen International, Inc., Gaithersburg, Md.;Cat. #IG50-0615, Lot #8077); HT Supplement 100× (GibcoBRL; Cat.#11067-030); HAT Supplement 100× (GibcoBRL; Cat. #31062-037); PEG 1500,(Boehringer Mannheim; Cat. #783 641); Red Blood Cell Lysing Buffer fromLab Services (Cat. #3KL-449); Trypane Blue; 70% Ethanol; Myeloma Cells(P3x) from ATCC.

The following equipment and supplies were used: laminar flow hood; CO₂incubator; inverted microscope; 37° C. water bath; centrifuge; 96-wellflat bottom tissue culture plates (Corning; Cat. #25860-96); Serologicalpipets and Integrid petri dishes (Falcon); 50 ml centrifuge tubes(Corning; Cat. #430921); 15 ml conical tubes (Falcon; Cat. #2096);autoclaved scissors and forceps; multichannel pipet; wide orifice 200 μlpipet tips (Denville Scientific, Inc., Metuchen, N.J.; Cat. #P1105-CP);sterile pipet tips (VWR, Buffalo Grove, Ill.; Cat. #53508-794).

HAT and HT medium were made as follows:

HAT Medium HT Medium IMDM 500 ml IMDM 500 ml L-Glutamine 2.5 mlL-Glutamine 2.5 ml Pen/Strep 5 ml Pen/Strep 5 ml HAT Supplement 5 ml HTSupplement 5 ml Origen Hy. Clon. F. 10% Final Origen Hy. Clon. F. 10%Final

Mice were given a final boost 3-4 days prior to fusion in PBSintravenously or intraperitoneal. Myeloma cells were kept in exponentialgrowth (log phase). The mice were euthanized, and the spleen from eachmouse was aseptically harvested. The spleen was placed in a petri dishcontaining warm Iscoves solution without FBS. A spleen cell suspensionwas prepared and transferred to a centrifuge tube. HAT-sensitive myelomacells were placed in a separate 50 ml centrifuge tube. Spleen cells andmyeloma cells were centrifuged at 400×g for 5 min, and the supernatantwas aspirated. The red blood cells from the spleen were lysed with 5 mlRBC Lysing Buffer for 1 min, and the tube was filled with SF media(hybridoma serum-free media; GibcoBRL; Cat. #12045-076). Splenocyteswere washed with 50 ml SF media, and spleen cells and myeloma cells werecentrifuged in separate centrifuge tubes at 400×g for 5 min. Spleencells and myeloma cells were counted and resuspended in 25 ml with SFmedia. Myeloma cells were added to spleen cells to give a 1:4 ratio. Themixture was centrifuged at 400×g for 10 min to form a tight pellet, andall media solution was removed by aspiration.

For the cell fusion experiments, PEG, SF media, and HAT media wereincubated at 37° C. One ml of 50% PEG was added to the cells for 1 min(PEG was added for 30 sec and cells were stirred for 30 sec). The PEGsolution was added to the side of the tube, and the pellet was gentlystirred. With stirring, 1 ml of SF media was added to the cells for 1min, and 8 ml of SF media was added to the cells for 2 min. Cells werecentrifuged at 400×g for 10 min, and the supernatant was aspirated.Cells were gently resuspended in 10 ml HAT selective media by aimingpipet directly at pellet and stirring. Additional HAT media was added tobring cell concentration to 5×10⁵ cells/ml. Cells were aliquotted into96-well tissue culture plates at 5×10⁴ cells/well. After 3 days, HTmedia was added at 100 μl/well, Approximately 10 days later, clones weretested for antibody production. Positive clones were expanded to 1 wellof 24-well plate. Positive clones were then re-tested, isotyped, andexpanded to T25 (0.25 cm square tissue culture flask).

ELISA Analysis:

To test for positive clones, ELISA analysis was performed using thefollowing reagents and supplies: carbonate/bicarbonate pH 9.6 (Sigma,St. Louis, Mo.; Cat. #C-3041) for coat; IMMULON® 2 ELISA plates (Dynex,Chantilly, Va.; Cat. #0110103455); 10×PBS (GibcoBRL) made to 1×concentration; wash buffer comprising TWEEN® 20 (0.05% finalconcentration) in 1×PBS; block buffer/sample diluent comprising washbuffer with 5% NFM non-fat milk; and chromogen mixture comprising 50%TMB (Kirkegaard & Perry Labs Gaithersburg, Md.; Cat. #50-76-01) and 50%peroxidase (Kirkegaard & Perry Labs Cat. #50-65-00).

For ELISA, plates were coated with 75 μl/well (1 μg/ml) BSL3-Ig incarbonate/bicarbonate overnight at 4° C. Plates were washed with PBSTWEEN° 20 (using Skatron), and blocked with 300 μl block buffer for 45min at room temperature. Plates were flicked dry and incubated with 75μl/well sera diluted in blocking buffer (sera was diluted 1:50 forhighest concentration and then serially diluted by factors of three) for45 min at room temperature, and washed as before. Plates were thenincubated with 75 μl/well anti-mouse IgG in blocking buffer (1:10000dilution; HRP-labeled; Amersham Pharmacia Biotech, Piscataway, N.J.) for45 min at room temperature, and washed as before. Following this, plateswere incubated with 100 μl/well chromogen mixture, and incubated up to15 min at room temperature. The signal was quenched with 100 μl 1Nsulfuric acid, and samples were read at 450/630 nm. Using ELISA,supernatants from hybridomas were initially screened against BSL3-Igfusion protein, and then screened against Ig protein alone. Hybridomasthat produced antibodies that bound to BSL3-Ig, but not Ig, weredesignated as positive clones.

Subcloning:

Positive clones from the initial fusion plate were expanded. Oncegrowing, cells were put through two rounds of single cell cloning toensure that they were monoclonal. Each hybridoma was plated in a 96-welltissue culture plate at a concentration of 0.5 cells/well or less. Oncemacroscopic colonies formed, supernatants were screened by ELISA.Positive clones from each hybridoma were titered by ELISA. The clonesgiving rise to the strongest signals were expanded and put through asecond round of cloning. Positive clonesNO: were again screened by ELISAand titered. The clones giving rise to the strongest signal wereexpanded and frozen back.

Example 7 Assays using BSL Monoclonal Antibodies

FACS Analysis of Lung Epithelial Cells Using BSL1 and BSL2 MonoclonalAntibodies:

A549, a lung epithelium cell line was cultured in RPM1 1640 (GibcoBRLCat. #11875-005) plus 10% FBS (Summit, Ft Collins, Colo.; Cat.#S-100-05) and 1% Penicillin-Streptomycin (GibcoBRL Cat. #15140-122) at37° C. and 5% CO₂ to 90% confluence in a T75 flask (Becton Dickinson,Franklin Lakes, N.J.; Cat. #353111). Cells were lifted with VERSENE®(GibcoBRL Cat. #15040-066) and washed twice in RPMI 1640. Cells wereadded to a 96-well plate (Becton Dickinson Cat. #353077) (2.5×10⁵ cellsper well), and centrifuged at 2000 RPM in a Beckman tabletop centrifuge.Next, cells were resuspended in RPMI 1640 or RPMI1640 with 1 μg negativecontrol antibody (MAb 15E10AA3) or hybridoma supernatant, and incubatedon ice for 30 min. Cells were then washed twice in RPMI 1640, andresuspended in goat anti-mouse anti-Fc FITC conjugated antibodies(BioSource, Camarillo Calif.; Cat. #AMI4408) diluted 1:50 in RPMI 1640.Following this, cells were incubated on ice for 30 min, washed twice inRPMI 1640, resuspended in RPMI 1640, and analyzed on a Becton DickinsonFACScan. FACS analysis indicated that BSL1 MAb 32F9A7 (FIG. 9A) andBSL2-4616811 (FIG. 9B) MAbs all bound to the A549 cells.

FACS Analysis of Various Cell Types Using BSL3 Monoclonal Antibodies:

To determine whether BSL3 polypeptide was expressed on the surface ofvarious cell types, cells were grown in media as shown in Table 7,below. HUT 78 was supplemented with 20% FBS (Summit Biotechnology, FtCollins, Colo.; Cat. #S-100-05). All other cell lines were supplementedwith 10% Summit FBS. In addition, all cell lines were supplemented with1% penicillin/streptomycin (GibcoBRL; Cat. #15140-122). All cell lineswere grown at 37° C. with 5% CO₂.

TABLE 7 CELL LINE ORIGIN MEDIA HL60 pre myeloid RPMI 1640 THP1 monocyticRPMI 1640 A549 lung epithelium RPMI 1640 H292 lung epithelium RPMI 1640PM LCL B lineage RPMI 1640 PL LCL B lineage RPMI 1640 CE LCL B lineageRPMI 1640 Ramos B lineage RPMI 1640 CEM T lineage RPMI 1640 HUT 78 Tlineage IMDM¹ Jurkat T lineage RPMI 1640² ¹IMDM: GibcoBRL; Cat.#12440-053 ²RPMI 1640: GibcoBRL; Cat. #11875-005

Cells were grown to about 5×10⁵ cells/ml. Cells were washed twice inserum free RPMI 1640 and resuspended to give a final concentration of2.5×10⁶ cells/ml. Anti-BSL3 MAb 1A4A1 antibodies or isotype control MAb15E10A3 antibodies were added to 5 μg/ml and incubated at 4° C. for 30min. Cells were washed twice in serum free RPMI 1640 and resuspended inserum free RPMI 1640 plus 2% goat anti-mouse IgG conjugated to FITC(BioSource, Camarillo, Calif.; Cat. #AMI4408). Cells were incubated 30min at 4° C. Cells were washed twice in serum free RPMI 1640 andanalyzed on a Becton Dickenson FACScan (Becton Dickenson, FranklinLakes, N.J.). BSL3 polypeptide was expressed on A549 (lung epithelium),H292 (lung epithelium), PM LCL (B lineage), PL LCL (B lineage), CE LCL(B lineage), and HUT78 (T lineage) cells (FIG. 9C).

FACS Analysis of Human Umbilical Vein Endothelial Cells Using BSL3Monoclonal Antibodies:

BSL3 monoclonal antibodies were used to measure BSL3 polypeptide levelsan human umbilical vein endothelial cells with or without TNF-alphastimulation. Human umbilical vein endothelial cells (HUVEC) were grownat 37° C. with 5% CO₂ in EGM media (Clonetics, Walkersville, Md.; Cat.#CC-4176) and grown to confluence. TNF-alpha was omitted or added to 10ng/ml for 24 hr. Cells were lifted with VERSENE® (GibcoBRL; Cat.#15040-066) and prepared for flow cytometry using anti-BSL3 MAb 1A4A1antibodies as described above. As a control, flow cytometry wasperformed without antibodies. The results indicated that BSL3polypeptide levels increased on TNF-alpha stimulated HUVEC (FIG. 9D).This increase was not observed in unstimulated cells (FIG. 9D).

FACS Analysis of Human Peripheral Blood Monocytes Using BSL3 MonoclonalAntibodies:

BSL3 monoclonal antibodies were used to measure BSL3 polypeptide levelson human peripheral blood monocytes with or without GM-CSF IL-4 or PHAstimulation. Peripheral blood monocytes (PBMCs) were purified asfollows. Blood samples were aliquotted equally among twelve 50 mlconical tubes. One volume of elutriation buffer (at room temperature)was added to each of the samples. Samples were underlayed with 10 ml LSM(lymphocyte separation mixture) ficoll solution, and centrifuged at 1800rpm for 25 min. The upper layer of reddish material was removed, and theLSM layer was transferred to a new tube (6 tubes per donor). Most of thePBMCs were observed on top of the LSM layer. Elutriation buffer (50 ml)was added to each tube, and the mixture was centrifuged at 1800 rpm for8 min. The supernatant was aspirated, and the PBMCs were resuspended in15 ml elutriation buffer. The mixture was transferred into 2 new 50 mlconical tubes. (45 ml total volume per tube), and centrifuged at 1000rpm for 8 min. The supernatant was aspirated, and this process wasrepeated two more times.

PBMCs were resuspended in flasks containing RPMI 1640 with 2% FBS, andincubated at 37° C. with 5% CO₂ for one hr. Flasks were rocked onceevery 20 min. Flasks were washed gently (twice) with media to removeT-cells and B cells. Flasks were then washed vigorously with RPMI 1640plus 10% FBS and 1% penicillin/streptomycin to obtain monocytes. PBMCswere washed twice in RPMI 1640 with 10% FBS and 1%penicillin/streptomycin. PBMCs were resuspended to 5×10⁶ cells/ml, andtransferred to flasks. PBMCs were incubated at 37° C. with 5% CO₂without stimulation for four days. In parallel experiments, PBMCs wereincubated with GM-CSF (15 ng/ml) and IL-4 (75 ng/ml) for four days, orPBMCs were incubated with PHA (1 μg/ml) for four days. Flasks werewashed vigorously with RPMI 1640 to remove the monocytes. PBMCs werewashed twice in RPMI 1640 examined by flow cytometry as described forthe various cell lines, above. The results indicated that BSL3polypeptide levels increased on GM-CSF IL4 or PHA stimulated cells(FIGS. 9E-9F). This increase was not observed in unstimulated cells(FIGS. 9E-9F).

Peripheral Blood T Cell Costimulation:

96-well plates (Becton Dickinson Cat. #353072) were coated with theindicated amount of anti-CD3 MAb G19.4 (described previously) in PBS(GibcoBRL Cat. #14190-144). Plates were incubated at 4° C. for 16 hr.Plates were washed twice in PBS. The following proteins were added:BSL2-4616811-Ig (20 μg/ml), BSL3-Ig (15 μg/ml), or Chi L6 (10 μg/ml) inPBS. Chi L6 is a protein fragment that comprises the Fc portion of humanIgG, and is identical to the Fc portion used in the BSL fusion proteins,described above. Different concentrations of protein were used to giveequivalent molarity. Plates were incubated at 37° C. for 4 hr. Plateswere washed twice in PBS. Peripheral blood T-cells were purified asdescribed, above. Cells were added (5×10⁴ cells per well) in RPMI with10% human serum (Sigma Cat. #H-4522) and 1% penicillin/streptomycin.Cells were incubated at 37° C. and 5% CO₂ for 72 hr. During the last 8hr, cells were incubated with an additional 50 μl of media containing 50μCi/ml ³H-thymidine (NEN Cat. #NET-027). Cells were harvested on aBrandel cell harvester (Brandel, Gaithersburg, Md.) using Packard GF/Cplates (Packard, Meriden, Conn.; Cat. #6005174), and the plates wereair-dried overnight. After this, 50 μl Microscint 20 (Packard Cat.#6013621) was added, and the radiolabel was counted on a PackardTopcount NXT.

Blockade of Peripheral Blood T Cell Costimulation Using BSL2 and BSL3Monoclonal Antibodies:

96-well plates (Becton Dickinson Cat. #353072) were coated with 20 μg/mlCD3 MAb G19.4 (previously described) in PBS (GibcoBRL Cat. #14190-144).Plates were incubated at 4° C. for 16 hr. Plates were washed twice inPBS. The following proteins were added: BSL2-4616811-Ig (20 μg/ml),BSL3-Ig (15 μg/ml), or (10 μg/ml) L6-Ig in PBS. Different concentrationsof protein were used to give equivalent molarity. Plates were incubatedat 37° C. for 4 hr. Plates were washed twice in PBS. Peripheral bloodT-cells were purified as described, above. Cells were added (5×10⁴ cellsper well) in RPMI with 10% human serum (Sigma Cat. #H-4522) and 1%GibcoBRL penicillin/streptomycin. Purified BSL2 or BSL3 MAbs or controlisotype MAbs were added to a final concentration of 20 μg/ml. To assayco-stimulation, MAbs were omitted. Plates were incubated at 37° C. and5% CO₂ for 72 hr. During the last eight hours, cells were incubated inan additional 50 μl of media with 50 μCi/ml ³H-thymidine (NEN Cat.#NET-027). The cells were harvested on a Brandel cell harvester usingPackard GF/C plates (Cat. #6005174) the plates were air-dried overnight.Following this, 50 μl Microscint 20 (Packard Cat. #6013621) was added,and the radiolabel was counted on a Packard Topcount NXT.

The results indicated that BSL2-4616811-Ig and BSL3-Ig fusion proteinsacted as co-stimulatory molecules for peripheral blood T-cells incubatedwith CD3 MAb G19.4 (FIGS. 10A-10F). This was confirmed with threeseparate peripheral blood T-cells donors: donor 010 (FIG. 10A); donor127 (FIG. 10B); donor 078 (FIGS. 10C-10D); and donor 124 (FIGS.10E-10F). The results further indicated that BSL2 and BSL3 MAbs blockedthe co-stimulatory effect of the BSL2-4616811-Ig and BSL3-Ig fusionproteins, respectively (FIGS. 10G-10J). This was confirmed with twoseparate peripheral blood T-cells donors: donor 010 (FIGS. 10G-10H); anddonor 127 (FIGS. 10I-10J).

As various changes can be made in the above compositions and methodswithout departing from the scope and spirit of the invention, it isintended that all subject matter contained in the above description,shown in the accompanying drawings, or defined in the appended claims beinterpreted as illustrative, and not in a limiting sense.

The contents of all patents, patent applications, published articles,books, reference manuals, texts and abstracts cited herein are herebyincorporated by reference in their entirety to more fully describe thestate of the art to which the present invention pertains.

What is claimed is:
 1. An isolated polypeptide comprising theextracellular domain of the BSL3 polypeptide provided as SEQ ID NO:15.2. An isolated polypeptide consisting of the extracellular domain of theBSL3 polypeptide provided as SEQ ID NO:15.
 3. An isolated polypeptideconsisting of between 170 and 200 contiguous amino acids of the BSL3polypeptide provided as SEQ ID NO:15.
 4. An isolated polypeptideaccording to claim 1, 2, or 3, fused to the Fc domain of human IgG.